KR20230045097A - Methods for Selective Cell-Free Nucleic Acid Analysis - Google Patents

Abstract

A method of processing a plurality of nucleic acid molecules derived from a cell-free biological sample by contacting the plurality of nucleic acid molecules or derivatives thereof with a plurality of binding agents, comprising: first sub-units of the plurality of nucleic acid molecules coupled to the plurality of binding agents; providing a set and a second subset of the plurality of nucleic acid molecules; separating the first subset of the plurality of nucleic acid molecules coupled to the plurality of binding agents from the second subset of the plurality of nucleic acid molecules; circularizing nucleic acid molecules derived from the first subset of the plurality of nucleic acid molecules to obtain circularized nucleic acid molecules; and identifying the circularized nucleic acid molecule or derivative thereof.

Description

Methods for Selective Cell-Free Nucleic Acid Analysis

cross reference

This patent application claims priority to U.S. Provisional Application No. 63/067,800, filed on August 19, 2020, which is hereby incorporated by reference in its entirety.

Nucleic acid variations often include differences in protein binding, such as nucleic acid binding proteins, including but not limited to transcription factors, as well as nucleic acid modifications, such as methylation. These variations are sometimes associated with a disease or risk of developing a disease. Thus, analysis of nucleic acids that bind to specific proteins or have specific alterations can be useful for disease diagnosis and treatment.

outline

In one aspect, provided herein is a method of processing a plurality of nucleic acid molecules derived from a cell-free biological sample. In some cases, the methods herein include (a) contacting a plurality of nucleic acid molecules or derivatives thereof with a plurality of binding agents to obtain a first subset of the plurality of nucleic acid molecules coupled to the plurality of binding agents and a second subset of the plurality of nucleic acid molecules coupled to the plurality of binding agents. providing a subset; (b) separating a first subset of the plurality of nucleic acid molecules coupled to the plurality of binding agents from a second subset of the plurality of nucleic acid molecules; (c) after (b), circularizing the nucleic acid molecules derived from the first subset of the plurality of nucleic acid molecules to obtain circularized nucleic acid molecules; and (d) identifying the circularized nucleic acid molecule or derivative thereof. In some cases, the plurality of nucleic acid molecules comprises deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. In some cases, circularization involves ligating together the 5' and 3' ends of the nucleic acid molecule. In some cases, circularization includes coupling the adapter to the 3' end, the 5' end, or both the 5' and 3' ends of the nucleic acid molecule. In some cases the method further comprises nucleic acid amplifying the circularized nucleic acid molecule to generate a plurality of amplification products of the circularized nucleic acid molecule, where (d) comprises identifying the plurality of nucleic acid amplification products. . In some cases, nucleic acid amplification is performed by a polymerase having strand displacement activity. In some cases, nucleic acid amplification is performed by a polymerase that does not have strand displacement activity. In some cases, nucleic acid amplification involves contacting circularized nucleic acid molecules with an amplification reaction mixture that includes random primers. In some cases, nucleic acid amplification involves contacting a circularized nucleic acid molecule with an amplification reaction mixture comprising one or more primers, each of which through sequence complementarity specifically hybridizes to a different target sequence. In some cases, binding agents include antibodies or fragments thereof. In some cases, the antibody or fragment thereof specifically binds to a nucleic acid binding protein. In some cases, the nucleic acid binding protein is a chromatin protein. In some cases, the nucleic acid binding protein is a histone. In some cases, the nucleic acid binding protein is a methyl CpG binding protein. In some cases, the nucleic acid binding protein is a transcription factor. In some cases, the nucleic acid binding protein is an RNA binding protein. In some cases, an antibody or fragment thereof specifically binds to a nucleic acid sequence. In some cases, nucleic acid sequences are methylated. In some cases, binding agents include polypeptides or nucleic acids. In some cases, the polypeptide includes streptavidin. In some cases the method further comprises determining the size of each cell-free nucleic acid molecule of the plurality of cell-free nucleic acid molecules. In some cases, (d) includes sequencing the circularized nucleic acid molecule or derivative thereof. In some cases, sequencing is sequencing by synthesis, sequencing by ligation, nanopore sequencing, nanoball sequencing, ion detection, sequencing by hybridization, polymerized colony (POLONY) sequencing, nanogrid rolling circle sequencing (ROLONY) and ion torrent sequencing. In some cases, a cell-free biological sample contains less than 75 nanograms of nucleic acids. In some cases, cell-free biological samples include bodily fluids. In some cases, the bodily fluid is urine, saliva, blood, serum, plasma, tears, sputum, cerebrospinal fluid, synovial fluid, mucus, bile, semen, lymph, amniotic fluid, menstrual fluid, or combinations thereof. In some cases, (d) includes sequencing the circularized nucleic acid molecule or derivative thereof. In some cases the method further comprises processing the sequences against the plurality of reference sequences to identify sequences corresponding to at least a subset of the plurality of reference sequences, thereby determining whether the subject has or is at risk of having the disease. do. In some cases, the disease is cancer. In some cases, the cancer consists of colon cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, hepatocellular carcinoma, liver cancer, skin cancer, malignant melanoma, endometrial cancer, esophageal cancer, gastric cancer, ovarian cancer, pancreatic cancer, brain cancer, leukemia, lymphoma, and myeloma. selected from the group. In some cases the method further comprises generating an electronic report indicating that the subject has or is at risk of having the disease using the identified sequence. In some cases, the method further comprises providing therapeutic intervention to a subject for a disease using the identified sequence. In some cases the method further comprises treating the subject for a disease using the identified sequence. In some cases, a subject is treated by subjecting the subject to chemotherapy or immunotherapy. In some cases the method further comprises monitoring the subject for progression or regression using the identified sequence. In some cases, (c) the nucleic acid molecule is coupled to a binding agent of a plurality of binding agents.

In another aspect, a method of processing a plurality of nucleic acid molecules derived from a cell-free biological sample, comprising: (a) determining the methylation status of the nucleic acid molecules of the plurality of nucleic acid molecules; (b) determining the size of the nucleic acid molecules of the plurality of nucleic acid molecules; and (c) processing (i) the methylation status of the nucleic acid molecules of the plurality of nucleic acid molecules for the first database, and (ii) the size of the nucleic acid molecules of the plurality of nucleic acid molecules for the second database, at least one disease A method comprising ascertaining the association of hypermethylation status and size is provided. In some cases, determining methylation status includes sequencing nucleic acid molecules of a plurality of nucleic acid molecules. In some cases, sequencing includes sequencing by synthesis, sequencing by ligation, nanopore sequencing, nanoball sequencing, ion detection, sequencing by hybridization, polymerized colony (POLONY) sequencing, nanogrid rolling circle sequencing (ROLONY), and ion detection. including a method selected from one or more of torrent sequencing. In some cases, determining the methylation status involves contacting the nucleic acid molecule with a binding agent that specifically binds to the methylated nucleic acid or derivative thereof. In some cases, determining the methylation status comprises (a) contacting the plurality of nucleic acid molecules with a plurality of binding agents, wherein a first subset of the plurality of nucleic acid molecules coupled to the plurality of binding agents and a second subset of the plurality of nucleic acid molecules are coupled to the plurality of binding agents. providing a subset; (b) separating a first subset of the plurality of nucleic acid molecules coupled to the plurality of binding agents from a second subset of the plurality of nucleic acid molecules; (c) after (b), circularizing the nucleic acid molecules derived from the first subset of the plurality of nucleic acid molecules to obtain circularized nucleic acid molecules; and (d) identifying the circularized nucleic acid molecule or derivative thereof. In some cases, binding agents include antibodies or fragments thereof. In some cases, binding agents include polypeptides or nucleic acids. In some cases, the polypeptide includes streptavidin. In some cases, the plurality of nucleic acid molecules comprises deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. In some cases, circularization involves ligating together the 5' and 3' ends of the nucleic acid molecule. In some cases, circularization includes coupling the adapter to the 3' end, the 5' end, or both the 5' and 3' ends of the nucleic acid molecule. In some cases the method further comprises nucleic acid amplifying the circularized nucleic acid molecule to generate a plurality of amplification products of the circularized nucleic acid molecule, where (d) comprises identifying the plurality of nucleic acid amplification products. . In some cases, nucleic acid amplification is performed by a polymerase having strand displacement activity. In some cases, nucleic acid amplification is performed by a polymerase that does not have strand displacement activity. In some cases, nucleic acid amplification involves contacting circularized nucleic acid molecules with an amplification reaction mixture that includes random primers. In some cases, nucleic acid amplification involves contacting a circularized nucleic acid molecule with an amplification reaction mixture comprising one or more primers, each of which through sequence complementarity specifically hybridizes to a different target sequence. In some cases, (d) includes sequencing the circularized nucleic acid molecule or derivative thereof. In some cases, sequencing includes sequencing by synthesis, sequencing by ligation, nanopore sequencing, nanoball sequencing, ion detection, sequencing by hybridization, polymerized colony (POLONY) sequencing, nanogrid rolling circle sequencing (ROLONY), and ion detection. including a method selected from one or more of torrent sequencing. In some cases, a cell-free biological sample contains less than 75 nanograms of nucleic acids. In some cases, cell-free biological samples include bodily fluids. In some cases, the bodily fluid is urine, saliva, blood, serum, plasma, tears, sputum, cerebrospinal fluid, synovial fluid, mucus, bile, semen, lymph, amniotic fluid, menstrual fluid, or combinations thereof. In some cases, the method processes a methylation state relative to a plurality of reference methylation states and processes a size relative to a plurality of reference sizes to obtain a methylation state corresponding to at least a subset of the plurality of reference methylation states and at least one subset of the reference size. and determining whether the subject has or is at risk of having the disease by ascertaining a size corresponding to the set. In some cases, the disease is cancer. In some cases, the cancer consists of colon cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, hepatocellular carcinoma, liver cancer, skin cancer, malignant melanoma, endometrial cancer, esophageal cancer, gastric cancer, ovarian cancer, pancreatic cancer, brain cancer, leukemia, lymphoma, and myeloma. selected from the group. In some cases, the method further includes generating an electronic report using the identified methylation status and size to indicate that the subject has or is at risk of having the disease. In some cases, the method further comprises providing therapeutic intervention to the subject for the disease using the identified methylation status and size. In some cases, the method further comprises treating the subject for a disease using the identified methylation status and size. In some cases, a subject is treated by subjecting the subject to chemotherapy or immunotherapy. In some cases, the method further comprises monitoring the subject for progression or regression using the identified methylation status and size.

In another aspect, a method of processing a plurality of nucleic acid molecules derived from a cell-free biological sample of a subject, comprising: (a) contacting a plurality of nucleic acid molecules or derivatives thereof with a plurality of binding agents, thereby providing a plurality of nucleic acid molecules coupled to the plurality of binding agents; providing a first subset of nucleic acid molecules and a second subset of a plurality of nucleic acid molecules; (b) separating the first subset of the plurality of nucleic acid molecules coupled to the plurality of binding agents from the second subset of the plurality of nucleic acid molecules; (c) after (b), circularizing the nucleic acid molecules derived from the first subset of the plurality of nucleic acid molecules to obtain a first subset of circularized nucleic acid molecules; and (d) after (b), circularizing nucleic acid molecules derived from the second subset of the plurality of nucleic acid molecules to obtain a second subset of circularized nucleic acid molecules; (e) sequencing a first subset of circularized nucleic acid molecules or derivatives thereof to obtain a first size and sequencing a second subset of circularized nucleic acid molecules or derivatives thereof to obtain a second size; (f) comparing the first magnitude and the second magnitude to determine the level of disease in the subject.

In another aspect, a method of processing a plurality of nucleic acid molecules derived from a cell-free biological sample of a subject, comprising: (a) contacting a plurality of nucleic acid molecules or derivatives thereof with a plurality of binding agents, thereby providing a plurality of nucleic acid molecules coupled to the plurality of binding agents; providing a first subset of nucleic acid molecules and a second subset of a plurality of nucleic acid molecules; (b) separating a first subset of the plurality of nucleic acid molecules coupled to the plurality of binding agents from a second subset of the plurality of nucleic acid molecules; (c) after (b), circularizing the nucleic acid molecules derived from the first subset of the plurality of nucleic acid molecules to obtain a first subset of circularized nucleic acid molecules; and (d) after (b), circularizing the nucleic acid molecules derived from the second subset of the plurality of nucleic acid molecules to obtain a second subset of circularized nucleic acid molecules; (e) sequencing a first subset of circularized nucleic acid molecules or derivatives thereof to obtain a first final sequence and sequencing a second subset of circularized nucleic acid molecules or derivatives thereof to obtain a second final sequence; step; (f) comparing the first final sequence and the second final sequence to determine the level of disease in the subject.

Citation by reference

All publications, patents and patent applications mentioned herein are incorporated herein by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

The novel features of the invention are pointed out with particularity in the appended claims. A better understanding of the features and advantages of the present invention may be obtained by reference to the following detailed description, which sets forth exemplary embodiments in which the principles of the present invention are utilized, and to the accompanying drawings (herein also referred to as "Figures" and "Figures"). will be obtained
1 schematically illustrates a computer system that may be programmed or otherwise configured to implement the methods of the present disclosure.

details

Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Numerous variations, modifications and substitutions may be made by those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used.

As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value determined by one of ordinary skill in the relevant art, depending on how the value is measured or determined, i.e., measured It may depend in part on the limitations of the system. For example, "about" can mean within 1 or more than 1 standard deviation, depending on the practice in the art. As another example, “about” can mean a range of 20% or less, 10% or less, 5% or less, or 1% or less of a given value. In the context of a biological system or process, the term "about" can mean within an order of magnitude, such as within 5-fold or within 2-fold of a value. Where specific values are recited in applications and claims, unless otherwise specified, the term "about" means within an acceptable error range for the specific value.

As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, generally deoxyribonucleotides (DNA) or ribonucleotides (RNA), or analogs thereof. A polynucleotide can be a nucleic acid molecule. A polynucleotide (or oligonucleotide) can have a sequence of nucleotides or nucleic acids. Polynucleotides can have any three-dimensional structure and can perform any function. The following are non-limiting examples of polynucleotides: cell-free nucleic acid, cell-free DNA (cfDNA), cell-free RNA (cfRNA), circulating tumor DNA (ctDNA), circulating tumor RNA (ctRNA), coding for a gene or gene fragment, or Non-coding regions, loci defined from linkage analysis (loci), exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA) ), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may include one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. A sequence of nucleotides may be interrupted by non-nucleotide elements. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

As used herein, the term "subject" generally refers to a vertebrate, such as a mammal (eg, a human). Mammals include, but are not limited to, murines, apes, humans, farm animals, sport animals, and pets (eg, dogs or cats). Tissues, cells, and progeny thereof of a biological subject obtained in vivo or cultured in vitro are also included. A subject may be a patient. A subject may have symptoms associated with a disease (eg, cancer). Alternatively, the subject may be asymptomatic in relation to the disease.

As used herein, the term "biological sample" generally refers to a sample derived from or obtained from a subject, such as a mammal (eg, a human). Biological samples include hair, fingernails, skin, sweat, tears, eye fluid, nasal swabs or nasopharyngeal lavage fluid, sputum, throat swabs, saliva, mucus, blood, serum, plasma, placental fluid, amniotic fluid, umbilical cord blood, umbilical cord fluid, cerebral fluid, earwax. , oil, glandular secretion, bile, lymph, pus, microbiome, meconium, breast milk, bone marrow, bone, CNS tissue, cerebrospinal fluid, adipose tissue, synovial fluid, stool, gastric fluid, urine, semen, vaginal secretion, stomach, small intestine, large intestine, rectum, pancreas, liver, kidney, bladder, lung, and other tissues and fluids derived from or obtained from a subject. A biological sample may be a cell-free (or cell-free) biological sample.

As used herein, the term “cell-free biological sample” generally refers to a sample derived from or obtained from a cell-free subject. A cell-free biological sample may include one or more cell-free molecules, such as cell-free DNA, cell-free RNA, or cell-free polypeptides (eg, proteins). Cell-free biological samples include blood, serum, plasma, nasal swab or nasopharyngeal lavage fluid, saliva, urine, gastric fluid, tears, feces, mucus, sweat, ear wax, oil, glandular secretions, bile, lymph, cerebrospinal fluid, tissue, semen, and vaginal fluid. , interstitial fluid, including interstitial fluid derived from tumor tissue, eye fluid, spinal cord, throat swab, breath, hair, nails, skin, biopsy, placental fluid, amniotic fluid, umbilical cord blood, spleen fluid, sinus fluid, sputum, pus, microflora, It may include, but is not limited to, meconium, human milk and/or other feces.

As used herein, the term "binding agent" generally refers to a substance that binds to a nucleic acid. Binding agents may include, but are not limited to, one or more of antibodies or fragments thereof, polypeptides, streptavidin, nucleic acids, DNA probes, or RNA probes.

As used herein, the terms “early cancer” and “non-metastatic cancer” generally refer to a cancer that may not metastasize in a subject (ie, the cancer has not left its initial location and spread to other locations). Early cancers can metastasize in a subject. Alternatively, an early cancer may not metastasize in a subject. The exact stage may depend on the type of cancer.

As used herein, the terms "tumor burden" and "tumor burden" generally refer to the size of a tumor or amount of cancer in a subject's body.

Whenever the terms "at least", "more than", or "at least" precede the first numerical value in a sequence of two or more numerical values, the terms "at least", "greater than", or "greater than" occur respectively in the sequence of numerical values. is applied to the numerical value of For example, 1, 2, or 3 or more is 1 or more, 2 or more, or 3 or more.

Whenever the terms "within", "less than", or "less than" precede the first numerical value in a sequence of two or more numerical values, the term "within", "less than" or "less than" occurs in the sequence of numerical values. Applies to each numerical value. For example, 3, 2, or 1 or less is 3 or less, 2 or less, or 1 or less.

Provided herein are methods, systems, and compositions for analyzing nucleic acids from a cell-free biological sample comprising a selection of at least a subset of the nucleic acids in the cell-free biological sample. Some of these methods include immunoprecipitation and sequence or size analysis of nucleic acids bound to nucleic acids, such as methylated nucleic acids or proteins, such as nucleic acid binding proteins (eg, transcription factors), using binding agents such as antibodies or fragments thereof; isolation of bound nucleic acids for further analysis, such as

Methods for Selective Nucleic Acid Analysis

Methods for selective nucleic acid analysis are provided herein. In some cases, a method of processing a plurality of nucleic acid molecules derived from a cell-free biological sample includes contacting the plurality of nucleic acid molecules with a plurality of binding agents, such as antibodies or fragments thereof. It provides a first subset of the plurality of nucleic acid molecules and a second subset of the plurality of nucleic acid molecules coupled to the plurality of antibodies or fragments thereof. Next, a first subset of the plurality of nucleic acid molecules coupled to the plurality of antibodies or fragments thereof can be separated from a second subset of the plurality of nucleic acid molecules. Next, the nucleic acid molecules derived from the first subset of the plurality of nucleic acid molecules can be circularized to obtain circularized nucleic acid molecules. The method then involves identifying the circularized nucleic acid molecule or derivative thereof.

In aspects of the methods for selective nucleic acid analysis provided herein, in some cases, the plurality of nucleic acid molecules comprises deoxyribonucleic acid (DNA) molecules. In some cases, the plurality of nucleic acid molecules includes ribonucleic acid (RNA) molecules. In some cases, the plurality of nucleic acid molecules includes a mixture of RNA molecules and DNA molecules.

In aspects of the methods for selective nucleic acid analysis provided herein, in some cases, circularization includes ligating together the 5' and 3' ends of the nucleic acid molecule. In some cases, circularization involves coupling the adapter to the 3' end, the 5' end, or both the 5' and 3' ends of the nucleic acid molecule.

In aspects of the methods for selective nucleic acid analysis provided herein, in some cases the method further comprises nucleic acid amplifying the circularized nucleic acid molecule to generate a plurality of amplification products of the circularized nucleic acid molecule, wherein the identification step includes identifying a plurality of nucleic acid amplification products. In some cases, nucleic acid amplification is performed by a polymerase having strand displacement activity. In some cases, nucleic acid amplification is performed by a polymerase that does not have strand displacement activity. In some cases, nucleic acid amplification involves contacting circularized nucleic acid molecules with an amplification reaction mixture comprising random primers. In some cases, nucleic acid amplification involves contacting a circularized nucleic acid molecule with an amplification reaction mixture comprising one or more primers, each of which through sequence complementarity specifically hybridizes to a different target sequence. In some cases, nucleic acid amplification involves contacting circularized nucleic acid molecules with an amplification mixture comprising one or more random primers.

In aspects of the methods for selective nucleic acid analysis provided herein, in some cases, a binding agent, such as an antibody or fragment thereof, specifically binds a nucleic acid. In some cases, the antibody or fragment thereof specifically binds to a nucleic acid binding protein. In some cases, the nucleic acid binding protein is a chromatin protein. In some cases, the nucleic acid binding protein is a histone. In some cases, the nucleic acid binding protein is a methyl CpG binding protein. In some cases, the nucleic acid binding protein is a transcription factor. In some cases, the nucleic acid binding protein is a polymerase. In some cases, the nucleic acid binding protein is a nuclease. In some cases, the nucleic acid binding protein includes a zinc finger motif, a helix-turn-helix motif, or a leucine zipper motif. In some cases, the nucleic acid binding protein is an RNA binding protein. In some cases, the nucleic acid binding protein is a splicing protein or RNA transport protein. In some cases, an antibody or fragment thereof specifically binds to a nucleic acid sequence. In some cases, an antibody or fragment thereof specifically binds to a methylated nucleic acid sequence. In some cases, an antibody or fragment thereof specifically binds to a phosphorylated nucleic acid sequence. In some cases, an antibody or fragment thereof specifically binds to an acetylated nucleic acid sequence. In some cases, a nucleic acid molecule is coupled to an antibody or fragment thereof of a plurality of antibodies or fragments thereof.

In aspects of the methods for selective nucleic acid analysis provided herein, in some cases the methods further include determining the size of each cell-free nucleic acid molecule of the plurality of cell-free nucleic acid molecules. In some cases, determining the size of each cell-free nucleic acid molecule includes sequencing, electrophoresis, or a combination thereof.

In aspects of the methods for selective nucleic acid analysis provided herein, in some cases, identifying the circularized nucleic acid comprises sequencing the circularized nucleic acid molecule or derivative thereof. In some cases, sequencing is sequencing by synthesis, sequencing by ligation, nanopore sequencing, nanoball sequencing, ion detection, sequencing by hybridization, polymerized colony (POLONY) sequencing, nanogrid rolling circle sequencing (ROLONY), and ion detection. including a method selected from one or more of torrent sequencing.

In aspects of the methods for selective nucleic acid analysis provided herein, in some cases, a cell-free biological sample comprises less than 100 nanograms of nucleic acid. In some cases, a cell-free biological sample contains less than 90 nanograms of nucleic acids. In some cases, a cell-free biological sample contains less than 80 nanograms of nucleic acids. In some cases, a cell-free biological sample contains less than 75 nanograms of nucleic acids. In some cases, a cell-free biological sample contains less than 70 nanograms of nucleic acids. In some cases, a cell-free biological sample contains less than 60 nanograms of nucleic acids. In some cases, a cell-free biological sample contains less than 50 nanograms of nucleic acid.

In aspects of the methods for selective nucleic acid analysis provided herein, in some cases, a cell-free biological sample comprises a bodily fluid. In some cases, the bodily fluid is urine, saliva, blood, serum, plasma, tears, sputum, cerebrospinal fluid, synovial fluid, mucus, bile, semen, lymph, amniotic fluid, menstrual fluid, or combinations thereof.

Methods for determining whether a subject has or is at risk of having a disease

Provided herein are methods of determining whether a subject has or is at risk of having a disease, comprising selective nucleic acid analysis. In some cases, some such methods contact a plurality of nucleic acid molecules with a plurality of binding agents, such as antibodies or fragments thereof, to form a first subset of the plurality of nucleic acid molecules coupled to the plurality of antibodies or fragments thereof and the plurality of nucleic acids. and providing a second subset of molecules. Next, in some cases the method comprises separating a first subset of the plurality of nucleic acid molecules coupled to the plurality of antibodies or fragments thereof from a second subset of the plurality of nucleic acid molecules. The method then includes circularizing nucleic acid molecules derived from the first subset of the plurality of nucleic acid molecules to obtain circularized nucleic acid molecules. The method then identifies the sequence of the circularized nucleic acid molecule or derivative thereof, processes the sequence against a plurality of reference sequences, and identifies sequences corresponding to at least a subset of the plurality of reference sequences, so that the subject has the disease. This includes determining whether or not there is a risk of In some cases, the plurality of nucleic acid molecules comprises deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. In some cases, a nucleic acid molecule is coupled to an antibody or fragment thereof of a plurality of antibodies or fragments thereof.

In aspects of the methods for selective nucleic acid analysis provided herein, in some cases, circularization includes ligating together the 5' and 3' ends of the nucleic acid molecule. In some cases, circularization includes coupling the adapter to the 3' end, the 5' end, or both the 5' and 3' ends of the nucleic acid molecule.

In aspects of the diagnostic methods provided herein, in some cases the method further comprises nucleic acid amplifying the circularized nucleic acid molecule to generate a plurality of amplification products of the circularized nucleic acid molecule, wherein the identifying step comprises a plurality of nucleic acid molecules. This includes identifying the amplification product. In some cases, nucleic acid amplification is performed by a polymerase having strand displacement activity. In some cases, nucleic acid amplification is performed by a polymerase that does not have strand displacement activity. In some cases, nucleic acid amplification involves contacting circularized nucleic acid molecules with an amplification reaction mixture comprising random primers. In some cases, nucleic acid amplification involves contacting a circularized nucleic acid molecule with an amplification reaction mixture comprising one or more primers, each of which through sequence complementarity specifically hybridizes to a different target sequence. In some cases, nucleic acid amplification involves contacting circularized nucleic acid molecules with an amplification mixture comprising one or more random primers.

In aspects of the diagnostic methods provided herein, in some cases, a binding agent, such as an antibody or fragment thereof, specifically binds a nucleic acid. In some cases, the antibody or fragment thereof specifically binds to a nucleic acid binding protein. In some cases, the nucleic acid binding protein is a chromatin protein. In some cases, the nucleic acid binding protein is a histone. In some cases, the nucleic acid binding protein is a methyl CpG binding protein. In some cases, the nucleic acid binding protein is a transcription factor. In some cases, the nucleic acid binding protein is a polymerase. In some cases, the nucleic acid binding protein is a nuclease. In some cases, the nucleic acid binding protein includes a zinc finger motif, a helix-turn-helix motif, or a leucine zipper motif. In some cases, the nucleic acid binding protein is an RNA binding protein. In some cases, the nucleic acid binding protein is a splicing protein or RNA transport protein. In some cases, an antibody or fragment thereof specifically binds to a nucleic acid sequence. In some cases, an antibody or fragment thereof specifically binds to a methylated nucleic acid sequence. In some cases, an antibody or fragment thereof specifically binds to a phosphorylated nucleic acid sequence. In some cases, an antibody or fragment thereof specifically binds to an acetylated nucleic acid sequence. In some cases, a nucleic acid molecule is coupled to an antibody or fragment thereof of a plurality of antibodies or fragments thereof.

In aspects of the diagnostic methods provided herein, in some cases the method further comprises determining the size of each cell-free nucleic acid molecule of the plurality of cell-free nucleic acid molecules. In some cases, determining the size of each cell-free nucleic acid molecule includes sequencing, electrophoresis, or a combination thereof.

In aspects of the diagnostic methods provided herein, in some cases, identifying the circularized nucleic acid comprises sequencing the circularized nucleic acid molecule or derivative thereof. In some cases, sequencing includes sequencing by synthesis, sequencing by ligation, nanopore sequencing, nanoball sequencing, ion detection, sequencing by hybridization, polymerized colony (POLONY) sequencing, nanogrid rolling circle sequencing (ROLONY), and ion detection. including a method selected from one or more of torrent sequencing.

In aspects of the diagnostic methods provided herein, in some cases, a cell-free biological sample comprises less than 100 nanograms of nucleic acid. In some cases, a cell-free biological sample contains less than 90 nanograms of nucleic acids. In some cases, a cell-free biological sample contains less than 80 nanograms of nucleic acids. In some cases, a cell-free biological sample contains less than 75 nanograms of nucleic acids. In some cases, a cell-free biological sample contains less than 70 nanograms of nucleic acids. In some cases, a cell-free biological sample contains less than 60 nanograms of nucleic acids. In some cases, a cell-free biological sample contains less than 50 nanograms of nucleic acids.

In aspects of the diagnostic methods provided herein, in some cases, a cell-free biological sample includes bodily fluid. In some cases, the bodily fluid is urine, saliva, blood, serum, plasma, tears, sputum, cerebrospinal fluid, synovial fluid, mucus, bile, semen, lymph, amniotic fluid, menstrual fluid, or combinations thereof.

In aspects of the diagnostic methods provided herein, in some cases the disease is cancer. In some cases, the cancer consists of colon cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, hepatocellular carcinoma, liver cancer, skin cancer, malignant melanoma, endometrial cancer, esophageal cancer, gastric cancer, ovarian cancer, pancreatic cancer, brain cancer, leukemia, lymphoma, and myeloma. selected from the group.

In aspects of the diagnostic methods provided herein, in some cases the method further comprises generating an electronic report indicating that the subject has or is at risk of having the disease using the identified sequence. In some cases, the method further comprises providing therapeutic intervention to a subject for a disease using the identified sequence. In some cases the method further comprises treating the subject for a disease using the identified sequence.

In aspects of the diagnostic methods provided herein, in some instances, a subject is treated by subjecting the subject to chemotherapy or immunotherapy.

In aspects of the diagnostic methods provided herein, in some cases the method further comprises monitoring the subject for progression or regression of the subject using the identified sequence.

Further provided herein are methods of determining whether a subject has or is at risk of having a disease comprising selective nucleic acid analysis herein. In some cases the method includes determining a methylation state for a nucleic acid molecule of a plurality of nucleic acid molecules and determining a size for a nucleic acid molecule of the plurality of nucleic acid molecules. The method then includes processing a methylation state for a nucleic acid molecule of a plurality of nucleic acid molecules against a first database, and processing a size for a nucleic acid molecule of a plurality of nucleic acid molecules against a second database. The method then includes determining whether the subject has or is at risk of having the disease, at least by ascertaining an association of methylation status and size with the disease.

In aspects of the diagnostic methods provided herein, in some cases the disease is cancer. In some cases, the cancer consists of colon cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, hepatocellular carcinoma, liver cancer, skin cancer, malignant melanoma, endometrial cancer, esophageal cancer, gastric cancer, ovarian cancer, pancreatic cancer, brain cancer, leukemia, lymphoma, and myeloma. selected from the group.

In aspects of the diagnostic methods provided herein, in some cases the methods include using the identified methylation status and size to generate an electronic report indicating that the subject has or is at risk of having the disease. In some cases, the method further comprises providing therapeutic intervention to the subject for the disease using the identified methylation status and size. In some cases, the method further comprises treating the subject for a disease using the identified methylation status and size. In some cases, a subject is treated by subjecting the subject to chemotherapy or immunotherapy. In some cases the method further comprises monitoring the subject for progression or regression using the identified methylation status and size.

Methylation and Size Nucleic Acid Analysis Methods

Methods for analyzing nucleic acids using methylation status and size are provided herein. In some cases the method includes determining a methylation state for a nucleic acid molecule of a plurality of nucleic acid molecules and determining a size for a nucleic acid molecule of the plurality of nucleic acid molecules. Next, the method includes processing a methylation state for a nucleic acid molecule of a plurality of nucleic acid molecules against a first database, and processing a size for a nucleic acid molecule of a plurality of nucleic acid molecules against a second database. The method then includes ascertaining an association of the size of the nucleic acid molecule and the methylation status of the nucleic acid molecule with at least one disease.

In aspects of the nucleic acid analysis methods herein, in some cases, determining methylation status includes sequencing nucleic acid molecules of a plurality of nucleic acid molecules. In some cases, sequencing is sequencing by synthesis, sequencing by ligation, nanopore sequencing, nanoball sequencing, ion detection, sequencing by hybridization, polymerized colony (POLONY) sequencing, nanogrid rolling circle sequencing (ROLONY), and ion detection. including a method selected from one or more of torrent sequencing. In some cases, determining methylation status includes bisulfite sequencing.

In aspects of the nucleic acid analysis methods herein, in some cases, determining methylation status comprises contacting a nucleic acid molecule with an antibody or fragment thereof that specifically binds to a methylated nucleic acid. In some cases, the antibody or fragment thereof specifically binds to a methylated nucleic acid binding protein or fragment thereof.

In aspects of the nucleic acid analysis methods herein, in some cases, determining methylation status comprises contacting a plurality of nucleic acid molecules with a plurality of antibodies or fragments thereof, thereby determining the number of nucleic acid molecules coupled to the plurality of antibodies or fragments thereof. providing one subset and a second subset of the plurality of nucleic acid molecules. The method then includes separating a first subset of the plurality of nucleic acid molecules coupled to the plurality of antibodies or fragments thereof from a second subset of the plurality of nucleic acid molecules. The method then includes circularizing nucleic acid molecules derived from the first subset of the plurality of nucleic acid molecules to obtain circularized nucleic acid molecules, and identifying circularized nucleic acid molecules or derivatives thereof.

In aspects of the nucleic acid analysis methods herein, in some cases, the plurality of nucleic acid molecules comprises deoxyribonucleic acid (DNA) molecules. In some cases, the plurality of nucleic acid molecules includes ribonucleic acid (RNA) molecules. In some cases, the plurality of nucleic acid molecules includes a mixture of RNA molecules and DNA molecules.

In aspects of the nucleic acid analysis methods herein, in some cases, circularization includes ligating together the 5' and 3' ends of the nucleic acid molecule. In some cases, circularization includes coupling the adapter to the 3' end, the 5' end, or both the 5' and 3' ends of the nucleic acid molecule.

In aspects of the nucleic acid analysis methods provided herein, in some cases the method further comprises nucleic acid amplifying the circularized nucleic acid molecule to generate a plurality of amplification products of the circularized nucleic acid molecule, wherein the identifying step comprises a plurality of It includes identifying the nucleic acid amplification product. In some cases, nucleic acid amplification is performed by a polymerase having strand displacement activity. In some cases, nucleic acid amplification is performed by a polymerase that does not have strand displacement activity. In some cases, nucleic acid amplification involves contacting circularized nucleic acid molecules with an amplification reaction mixture comprising random primers. In some cases, nucleic acid amplification involves contacting a circularized nucleic acid molecule with an amplification reaction mixture comprising one or more primers, each of which through sequence complementarity specifically hybridizes to a different target sequence. In some cases, nucleic acid amplification involves contacting circularized nucleic acid molecules with an amplification mixture comprising one or more random primers.

In aspects of the nucleic acid analysis methods herein, in some cases, an antibody or fragment thereof specifically binds to a nucleic acid. In some cases, the antibody or fragment thereof specifically binds to a nucleic acid binding protein. In some cases, the nucleic acid binding protein is a chromatin protein. In some cases, the nucleic acid binding protein is a histone. In some cases, the nucleic acid binding protein is a methyl CpG binding protein. In some cases, the nucleic acid binding protein is a transcription factor. In some cases, the nucleic acid binding protein is an RNA binding protein. In some cases, an antibody or fragment thereof specifically binds to a nucleic acid sequence. In some cases, an antibody or fragment thereof specifically binds to a methylated nucleic acid sequence. In some cases, a nucleic acid molecule is coupled to an antibody or fragment thereof of a plurality of antibodies or fragments thereof.

In aspects of the nucleic acid analysis methods herein, in some cases the method further comprises determining the size of each cell-free nucleic acid molecule of the plurality of cell-free nucleic acid molecules.

In aspects of the nucleic acid analysis methods herein, in some cases, identifying a circularized nucleic acid comprises sequencing the circularized nucleic acid molecule or derivative thereof. In some cases, sequencing is sequencing by synthesis, sequencing by ligation, nanopore sequencing, nanoball sequencing, ion detection, sequencing by hybridization, polymerized colony (POLONY) sequencing, nanogrid rolling circle sequencing (ROLONY), and ion detection. including a method selected from one or more of torrent sequencing.

In aspects of the nucleic acid analysis methods herein, in some cases, a cell-free biological sample comprises less than 100 nanograms of nucleic acid. In some cases, a cell-free biological sample contains less than 90 nanograms of nucleic acids. In some cases, a cell-free biological sample contains less than 80 nanograms of nucleic acids. In some cases, a cell-free biological sample contains less than 75 nanograms of nucleic acids. In some cases, a cell-free biological sample contains less than 70 nanograms of nucleic acids. In some cases, a cell-free biological sample contains less than 60 nanograms of nucleic acids. In some cases, a cell-free biological sample contains less than 50 nanograms of nucleic acids.

In aspects of the nucleic acid analysis methods herein, in some cases, a cell-free biological sample comprises a bodily fluid. In some cases, the bodily fluid is urine, saliva, blood, serum, plasma, tears, sputum, cerebrospinal fluid, synovial fluid, mucus, bile, semen, lymph, amniotic fluid, menstrual fluid, or combinations thereof.

System and Computer Assisted Methods

Systems for the selective nucleic acid analysis methods provided herein are provided herein. In some cases, the systems herein contact a computer configured to receive a user request to process a plurality of nucleic acid molecules derived from a cell-free biological sample and a plurality of nucleic acid molecules by contacting a plurality of binding agents, such as antibodies or fragments thereof, with a plurality of nucleic acid molecules. A processing unit for obtaining a first subset of the plurality of nucleic acid molecules and a second subset of the plurality of nucleic acid molecules coupled to an antibody of or a fragment thereof. In some cases, the system further comprises an isolation unit for separating a first subset of the plurality of nucleic acid molecules coupled to the plurality of antibodies or fragments thereof from a second subset of the plurality of nucleic acid molecules. In some cases, the system further comprises a circularization unit for circularizing nucleic acid molecules derived from the first subset of the plurality of nucleic acid molecules to obtain circularized nucleic acid molecules. In some cases, the system further comprises an identification unit for identifying the circularized nucleic acid molecule or derivative thereof. In some cases, the system further comprises a report generator that sends a report containing the identity of the circularized nucleic acid molecule or derivative thereof to the recipient.

A computer for use in the system may include one or more processors. A processor may be combined with one or more controllers, computational units, and/or other units of a computer system, or may be implemented in firmware as desired. When executed in software, the routines may be stored in any computer readable memory, such as RAM, ROM, flash memory, magnetic disk, laser disk, or other suitable storage medium. Likewise, such software may be delivered by any known method of delivery including, for example, over a communication channel such as a telephone line, the Internet, a wireless connection, or the like, or via a transferable medium such as a computer readable disk, a flash drive, or the like. It can be transmitted to the computing device through. The various steps may be implemented as various blocks, tasks, tools, modules and techniques, which in turn may be implemented as hardware, firmware, software, or any combination of hardware, firmware, and/or software. When executed in hardware, some or all of the blocks, tasks, techniques, etc. may be, for example, user integrated circuits (ICs), application specific integrated circuits (ASICs), field programmable logic arrays (FPGAs), programmable logic arrays. (PLA), etc. A client-server, relational database architecture may be used in embodiments of the system. A client-server architecture is a network architecture in which each computer or process on the network is either a client or a server. Server computers are typically powerful computers dedicated to managing disk drives (file servers), printers (print servers), or network traffic (network servers). Client computers include PCs (personal computers) or workstations on which users run applications as well as exemplary output devices as disclosed herein. Client computers depend on server computers for resources, such as files, devices, and even processing power. In some embodiments, the server computer handles all database functions. The client computer may have software that handles all front-end data management, and may also receive input data from the user.

The system can be configured to receive a user request to perform a detection reaction on a sample. User requests can be direct or indirect. Examples of direct requests include those sent by an input device, such as a keyboard, mouse, or touch screen. Examples of indirect requests include transmission over a communication medium, such as the Internet (wired or wireless).

The system may further include an amplification system that performs a nucleic acid amplification reaction on the sample or a portion thereof in response to a user request. A variety of methods are available for amplifying polynucleotides (eg, DNA and/or RNA). Amplification can be linear, exponential, or include both linear and exponential phases in a multi-step amplification process. The amplification method may involve a change in temperature, such as a heat denaturation step, or may be an isothermal process that does not require heat denaturation. Non-limiting examples of suitable amplification processes are described herein, as for any of the various aspects of the present disclosure. In some embodiments, amplification comprises rolling circle amplification (RCA). A variety of systems are available for amplifying polynucleotides, which may vary based on the type of amplification reaction being performed. For example, in the case of an amplification reaction involving cycles of temperature change, the amplification system may include a thermocycler. Amplification systems can include real-time amplification and detection instruments, such as systems manufactured by Applied Biosystems, Roche, and Stratagene. In some embodiments, the amplification reaction comprises (i) circularizing individual polynucleotides to form a plurality of circular polynucleotides, each having a junction between the 5' and 3' ends; and (ii) amplifying the circular polynucleotide. Samples, polynucleotides, primers, polymerases, and other reagents can be, eg, any of those described herein, for any of the various aspects. circularization process (e.g., with and without adapter oligonucleotides), reagents (e.g., type of adapter, use of ligase), reaction conditions (e.g., favoring self-ligation), Non-limiting examples of optional additional processes (eg, post-reaction purification), and junctions formed thereby, are provided herein, eg, in connection with any of the various aspects of the present disclosure. A system may be selected and/or designed to perform any of the above methods.

The system generates sequencing reads for polynucleotides amplified by the amplification system, identifies sequence differences between the sequencing reads and a reference sequence, and determines sequence differences present in at least two circular polynucleotides with different junctions as sequence variants. It may further include a sequencing system that does. The sequencing system and amplification system may include identical or overlapping equipment. For example, both an amplification system and a sequencing system can use the same thermocycler. A variety of sequencing platforms are available for use in the system and can be selected based on the sequencing method selected. Examples of sequencing methods are described herein. Amplification and sequencing may involve the use of a liquid handler. Several commercially available liquid handling systems can be used to implement the automation of these processes (eg from Perkin-Elmer, Beckman Coulter, Caliper Life Sciences, Tecan, Eppendorf, Apricot Design, Velocity 11 as examples). See Liquid Handler in ). A variety of automated sequencing instruments are commercially available and manufactured by Life Technologies (SOLiD platform, and pH-based detection), Roche (454 platform), Illumina (e.g., flow cytometry based systems such as genomic analyzer devices). contains a sequencer. Transfer between two, three, four, five or more automated devices (eg, between one or more liquid handling devices and a sequencing device) may be manual or automated.

The system may further include a report generator that sends a report to a recipient, where the report includes detection results of the sequence variant. Reports can be generated in real time, eg, during sequencing reads or as sequencing data is analyzed, with periodic updates as the process progresses. Additionally, or alternatively, a report may be generated at the conclusion of the analysis. A report can be automatically generated when such a sequencing system completes the steps of identifying intact nucleic acids. In some embodiments, a report is generated in response to instructions from a user. In addition to the results of identification of the nucleic acids, the report may also include an analysis based on the identified nucleic acids. For example, if one or more sequence variants are associated with a particular contaminant or phenotype, the report may include information related to this association, such as the likelihood that the contaminant or phenotype is present at some level, and optionally suggestions based on such information (e.g. For example, additional testing, monitoring, or remedial action). Reports can take any of a variety of forms. Data related to this disclosure may be used to transmit information, including but not limited to, mailing a physical report, e.g., a printout, to the network or connection (or for review by a recipient) for receipt and/or review by the recipient. It is envisioned that it can be transmitted by any other suitable means). The recipient can be, but is not limited to, a person or an electronic system (eg, one or more computers, and/or one or more servers).

Machine-readable media containing computer-executable code may take many forms, including but not limited to tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks, eg, any storage device in any computer, and the like, and may be used, eg, to run a database or the like. Volatile storage media include dynamic memory, such as the main memory of such computer platforms. Tangible transmission media include coaxial cable; Included are copper wires and optical fibers, including wires that contain buses in computer systems. Carrier-wave transmission media may take the form of electrical or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tapes, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punches Card stock tape, any other physical storage medium with a pattern of holes, RAM, ROM, PROM and EPROM, FLASH-EPROM, any other memory chip or cartridge, carrier waves carrying data or instructions, cables carrying such carrier waves, or links, or any other medium from which a computer can read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

The subject computer-executable code may be executed on any suitable device comprising a processor, including a server, PC, or mobile device such as a smartphone or tablet. Any controller or computer optionally includes a monitor, which may be a cathode ray tube (“CRT”) display, a flat panel display (eg, an active matrix type liquid crystal display, liquid crystal display, etc.), or the like. Computer circuitry is often enclosed in a box, which contains a number of integrated circuit chips, such as microprocessors, memory, interface circuitry, and the like. The box also optionally includes a hard disk drive, a floppy disk drive, a high capacity removable drive such as a recordable CD-ROM, and other common peripheral elements. An input device, such as a keyboard, mouse, or touch-sensitive screen, optionally provides input from the user. The computer is configured to receive user instructions in the form of user input into a set of parameter fields, eg, a GUI, or in the form of preprogrammed instructions, eg, preprogrammed instructions for a variety of different specific tasks. Appropriate software may be included.

Library preparation and amplification methods

The methods herein include, in certain instances, amplification of polynucleotides present in a sample from a subject. Amplification methods used herein often include rolling-circle amplification. Alternatively or in combination, amplification methods used herein include PCR. In some cases, amplification methods herein include linear amplification. Often, amplification is not targeted to one gene or set of genes and the entire nucleic acid sample is amplified. In some cases the method comprises (a) circularizing a plurality of individual polynucleotides to form a plurality of circular polynucleotides, each having a junction between a 5' end and a 3' end; and (b) amplifying the circular polynucleotide of (a) to produce an amplified polynucleotide. In a further case, the amplification method comprises (c) shearing the amplified polynucleotides to produce sheared polynucleotides, each sheared polynucleotide having one or more shear points at its 5' end and/or 3' end. Including, including steps. In some cases the method does not include enrichment for the target sequence.

Generally, joining the ends of polynucleotides together to form circular polynucleotides (either directly or using one or more intermediate adapter oligonucleotides) creates junctions having junction sequences. When the 5' end and 3' end of a polynucleotide are joined via an adapter polynucleotide, the term "junction" may refer to a junction between a polynucleotide and an adapter (e.g., a 5' end junction or a 3' end junction). one of the junctions), the junction between the 5' and 3' ends of a polynucleotide, such as formed by and including an adapter polynucleotide. Where the 5' and 3' ends of a polynucleotide are joined without intervening adapters (e.g., the 5' and 3' ends of single-stranded DNA), the term "junction" refers to the point at which these two ends are joined. do. A junction can be identified by a sequence of nucleotides comprising the junction (also referred to as “junction sequence”).

Samples herein are subject to natural degradation processes (e.g., cell lysis, cell death, and release of DNA and RNA from cells into their surroundings, e.g., cell-free polynucleotides, e.g., cell-free DNA and cell-free RNA). fragmentation as a by-product of sample processing (e.g., fixation, staining, and/or storage), and fragmentation by methods that cut DNA without restriction to a specific target sequence (e.g., eg by mechanical fragmentation, eg by sonication; by non-sequence specific nuclease treatment, eg DNase I, a fragmentase). If the sample contains polynucleotides with a mixture of ends, it is unlikely that two polynucleotides will have the same 5' end or 3' end, and the two polynucleotides will independently have both the same 5' and 3' ends. The odds of having them all are lower. Thus, in some embodiments, junctions can be used to distinguish different polynucleotides, even when the two polynucleotides contain a portion with the same target sequence. Where polynucleotide ends are ligated without intervening adapters, junction sequences can be identified by alignment to a reference sequence. For example, if the order of two component sequences appears to be reversed relative to the reference sequence, the point at which the inversion appears to occur may indicate a junction at that point. When polynucleotide ends are joined via one or more adapter sequences, the junctions are either by proximity to known adapter sequences, or sequences from both the 5' and 3' ends of the polynucleotide to which the sequencing reads were circularized as above. can be checked by alignment if it is of sufficient length to obtain In some embodiments, the formation of a particular junction is a sufficiently rare event that it is unique among the circularized polynucleotides of the sample.

In some embodiments, circularization of individual polynucleotides is achieved by subjecting a plurality of polynucleotides to a ligation reaction. A ligation reaction may involve a ligase enzyme. In some cases, the ligase enzyme is a single-stranded DNA or RNA ligase. In some cases, the ligase enzyme is a double-stranded DNA ligase. In some embodiments, the ligase enzyme is digested prior to amplification in (b). In (b), degradation of the ligase prior to amplification may increase the recovery rate of amplifiable polynucleotides. In some embodiments, the plurality of circularized polynucleotides are not purified or isolated prior to (b). In some embodiments, linear polynucleotides that are not circularized are digested prior to amplification. In some cases, the plurality of polynucleotides are denatured prior to circularization to create single-stranded polynucleotides; In some cases, the plurality of polynucleotides are not denatured prior to circularization.

In some cases, circularization in (a) includes linking and adapting the polynucleotides to the 5' end, 3' end, or both the 5' and 3' ends of the polynucleotides in the plurality of polynucleotides. As described above, when the 5' end and/or 3' end of a polynucleotide is joined via an adapter polynucleotide, the term "junction" may refer to a junction between a polynucleotide and an adapter (e.g., 5 'end junction or 3' end junction), a junction between the 5' and 3' ends of a polynucleotide, such as formed by and including an adapter polynucleotide.

Circularized polynucleotides are in some cases amplified, eg, after digestion with a ligase enzyme, to produce amplified polynucleotides. Amplification of circular polynucleotides can be performed by polymerases. In some cases, the polymerase is a polymerase with strand displacement activity. In some cases, the polymerase is Phi29 DNA polymerase. Alternatively, the polymerase is a polymerase that does not have strand displacement activity. In some cases, the polymerase is T4 DNA polymerase or T7 DNA polymerase. Alternatively or in combination, the polymerase is a Taq polymerase, or a polymerase of the Taq polymerase family. In some cases, the polymerase is a reverse transcriptase. In some cases, amplification includes rolling circle amplification (RCA). Amplified polynucleotides generated from RCA may include linear concatemers, or polynucleotides comprising more than one copy of a target sequence (eg, a subunit sequence) from a template polynucleotide. In some embodiments, amplification comprises applying circular polynucleotides to an amplification reaction mixture comprising random primers. In some cases, amplification involves applying the circular polynucleotide to an amplification reaction mixture comprising one or more primers, each of which specifically hybridizes to a different target sequence through sequence complementarity. In some cases, amplification includes applying the circular polynucleotide to an amplification reaction mixture comprising reverse primers.

Amplified polynucleotides are, in some cases, sheared to produce sheared polynucleotides that are shorter in length than unsheared polynucleotides. Two or more sheared polynucleotides from the same linear concatemer may have the same splice sequence, but may have different 5' and/or 3' ends (eg, shear ends).

Cell-free polynucleotides from the sample may be DNA, RNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro RNA (miRNA), messenger RNA (mRNA), small interfering RNA (siRNA), fragments of any of these, or any of a variety of polynucleotides including, but not limited to, combinations of any two or more of these. In some embodiments, a sample comprises DNA. In some embodiments, a sample comprises cell free genomic DNA. In some embodiments, a sample is amplified by amplification, such as a primer extension reaction using any suitable combination of DNA polymerase and primers, including but not limited to polymerase chain reaction (PCR), reverse transcription, and combinations thereof. contains the generated DNA. When the template for the primer extension reaction is RNA, the product of reverse transcription is referred to as complementary DNA (cDNA). Primers useful in the primer extension reaction can include one or more target-specific sequences, random sequences, partially random sequences, and combinations thereof. Generally, the sample polynucleotide includes any polynucleotide present in the sample, which may or may not include the target polynucleotide. A polynucleotide may be single-stranded, double-stranded, or a combination thereof. In some embodiments, the polynucleotides subjected to the method of disclosure are single-stranded polynucleotides that may or may not be present in the presence of double-stranded polynucleotides. In some embodiments, a polynucleotide is single-stranded DNA. Single-stranded DNA (ssDNA) may be ssDNA isolated in single-stranded form, or DNA isolated in double-stranded form and then made single-stranded for the purpose of one or more steps in the disclosed method.

In some embodiments, the polynucleotide is subjected to subsequent steps (eg, circularization and amplification) without an extraction step and/or without a purification step. For example, a fluid sample can be treated to remove cells without an extraction step to produce a purified liquid sample and a cell sample, and then DNA is isolated from the purified fluid sample. A variety of procedures are available for isolation of polynucleotides, such as precipitation or non-specific binding to a substrate followed by washing the substrate to release the bound polynucleotide. When polynucleotides are isolated from a sample without a cell extraction step, the polynucleotides will be primarily extracellular or "cell-free" polynucleotides, such as cell-free DNA and cell-free RNA, which may correspond to dead or damaged cells. The identity of such cells may be tumor cells (eg, in cancer detection), fetal cells (eg, in prenatal diagnosis), cells from transplanted tissue (eg, in early detection of transplant failure), or microbes. It can be used to characterize the cell or population of cells from which they are derived, such as members of a population.

When a sample is processed to extract polynucleotides, eg, from cells within the sample, a variety of extraction methods are available. For example, nucleic acids can be purified by organic extraction using phenol, phenol/chloroform/isoamyl alcohol, or similar formulations including TRIzol and TriReagent. Other non-limiting examples of extraction techniques include: (1) with or without an automated nucleic acid extractor, e.g., the Model 341 DNA extractor available from Applied Biosystems (Foster City, Calif.), e.g., phenol / organic extraction using chloroform organic reagent (Ausubel et al., 1993, incorporated herein by reference in its entirety) followed by ethanol precipitation; (2) the fixed bed adsorption method (U.S. Patent No. 5,234,809; Walsh et al., 1991, each of which is incorporated herein by reference in its entirety); and (3) salt-induced nucleic acid precipitation methods (Miller et al., (1988), incorporated herein by reference in its entirety), typically referred to as "salting out" methods. Another example of nucleic acid isolation and/or purification is the use of magnetic particles capable of specifically or non-specifically binding to nucleic acids, followed by isolating beads using a magnet, washing, and eluting the nucleic acids from the beads. (See, eg, US Pat. No. 5,705,628, incorporated herein by reference in its entirety). In some embodiments, the isolation method may proceed after an enzymatic digestion step to assist in the removal of unwanted proteins from the sample, eg, digestion with proteinase K, or other similar proteases. See, eg, US Pat. No. 7,001,724, incorporated herein by reference in its entirety. If desired, an RNase inhibitor may be added to the lysis buffer. For certain cell or sample types, it may be desirable to add a protein denaturation/degradation step to the protocol. Purification methods may relate to isolating DNA, RNA, or both. If both DNA and RNA are isolated together during or after the extraction procedure, additional steps may be used to separate and purify one or both from the others. Sub-fractions of extracted nucleic acids may also be generated by purification, for example by size, sequence, or other physical or chemical characteristics. In addition to the initial nucleic acid isolation step, purification of the nucleic acid may be performed after any step of the disclosed methods, such as to remove excess or unwanted reagents, reactants, or products. Various methods for determining the amount and/or purity of nucleic acids in a sample, such as absorbance (eg, absorbance of light at 260 nm, 280 nm, and ratios thereof) and detection of labels (eg, fluorescent dyes) and intercalating agents such as SYBR green, SYBR blue, DAPI, propidium iodide, Hoechst staining, SYBR gold, ethidium bromide).

In some cases, the methods herein include the preparation of DNA libraries from polynucleotides. For example, the methods herein include the preparation of single-stranded DNA libraries. Any suitable method for preparing single-stranded DNA libraries can be used in the methods herein. For example, a method of preparing a single-stranded DNA library includes generating a plurality of ssDNAs by denaturing a DNA sample; ligating an adapter to the 3' end of the ssDNA molecule or extending the 3' end of the ssDNA molecule through non-template synthesis; synthesizing a second strand using a primer complementary to the adapter or 3' extended sequence; ligating the double stranded adapter to the extension product; amplifying the second strand using primers targeting the first and second adapters (eg, using PCR); and sequencing the library on a sequencer. Additional methods of single-stranded library preparation include denaturing a DNA sample to generate a plurality of ssDNA; ligating the adapter to the 3' end of the ssDNA molecule; synthesizing a second strand using a primer complementary to the adapter; ligating the double stranded adapter to the extension product; amplifying the second strand using primers targeting the first and second adapters (eg, by PCR); selectively enriching for a region of interest using hybridization with a capture probe; amplifying the captured product (eg, by PCR); and sequencing the library on a sequencer.

Additional examples of single-stranded library preparation are described in Gansauge et al. 2013. Nature Protocols. 8(4) 737-748; treating the DNA with a heat labile phosphatase to remove residual phosphate groups from the 5' and 3' ends of the DNA strand; removing deoxyuracil derived from cytosine deamination from the DNA strand; ligating a 5'-phosphorylated adapter oligonucleotide having about 10 nucleotides and a long 3' biotinylated spacer arm to the 3' end of the DNA strand; immobilizing adapter-ligated molecules on streptavidin beads; copying the template strand using a 5'-tailed primer complementary to the adapter using Bst polymerase; washing off excess primer; removing the 3' overhang using T4 DNA polymerase; joining the second adapter to the newly synthesized strand using blunt-end ligation; washing excess adapters; releasing library molecules by thermal denaturation; Adding a full-length adapter sequence including a barcode through amplification using a tailed primer; and methods comprising sequencing the library.

In a further embodiment, the methods herein include the preparation of a double-stranded DNA library. Any suitable method of making a double-stranded DNA library can be used in the methods herein. For example, a method of making a double-stranded DNA library includes ligating sequencing adapters to the 5' and 3' ends of a plurality of DNA fragments and sequencing the library on a sequencer. A further method of preparing a double-stranded DNA library includes ligating adapters to the 5' and 3' ends of a plurality of DNA fragments; attaching the entire adapter sequence to the ligated fragment via PCR using a primer complementary to the ligated adapter; and sequencing the library on a sequencer. Additional methods include ligating adapters to the 5' and 3' ends of the plurality of DNA fragments; Amplifying the ligated product through PCR complementary to the ligated adapter; optionally enriching the region of interest through hybridization with a capture probe; PCR amplifying the captured product; and sequencing the library on a sequencer. Additional methods of double-stranded library preparation include ligating adapters to the 5' and 3' ends of a plurality of DNA fragments; amplifying the ligated product through PCR using a primer complementary to the ligated adapter; circularizing double-stranded PCR products or denaturing and circularizing single-stranded PCR products; enriching the region of interest by PCR using primers that selectively target specific genes; and sequencing the library on a sequencer.

Additional examples of double-stranded library preparation are described in Kinde et al., Kinde et al. 2011. Proc. Natl. Acad. Sci., USA, 108(23) 9530-9535], which assigns each template molecule a unique identifier (UID); amplification of each uniquely tagged template molecule to create a UID family; and duplicate sequencing of amplification products. Additional examples may be found in Lv et al., Lv et al. 2015. Clin. Chem., 61(1) 172-181], wherein the tag tags a single molecule with a unique barcode, circularizes it, and reverse PCR After targeting the allele for replication, the prepared library is sequenced and the alleles present are counted.

In an additional library construction method, cfDNA fragments with specific characteristics are selected using antibodies. In some cases, methylated or hypermethylated cfDNA fragments are selected using antibodies. The selected cfDNA fragments are then used in any of the library preparation methods described herein, including circularization, single-stranded DNA library preparation, and double-stranded DNA library preparation. Sequencing of these isolated cfDNA fragments provides information about features present in cfDNA, including modifications such as methylation or hypermethylation.

According to some embodiments, the polynucleotides in the plurality of polynucleotides from the sample are circularized. Circularization can be done by linking the 5' end of a polynucleotide to the 3' end of the same polynucleotide, to the 3' end of another polynucleotide in the sample, or to a polynucleotide from a different source (e.g., an artificial polynucleotide) to the 3' end. In some embodiments, the 5' end of a polynucleotide is joined to the 3' end of the same polynucleotide (also referred to as "self-joining"). In some embodiments, the conditions of the circularization reaction are selected to favor self-association of polynucleotides within a particular range of lengths to produce a population of circularized polynucleotides of a particular average length. For example, circularization reaction conditions may be selected to favor self-association of polynucleotides shorter than about 5000, 2500, 1000, 750, 500, 400, 300, 200, 150, 100, 50 or less nucleotides in length. can In some embodiments, fragments having a length of 50 to 5000 nucleotides, 100 to 2500 nucleotides, or 150 to 500 nucleotides are advantageous such that the average length of the circularized polynucleotides falls within the respective ranges. In some embodiments, at least 80% of the circularized fragments are between 50 and 500 nucleotides in length, such as between 50 and 200 nucleotides in length. Reaction conditions that can be optimized include the length of time allotted for the ligation reaction, the concentrations of various reagents, and the concentration of polynucleotides to be ligated. In some embodiments, the circularization reaction preserves the distribution of fragment lengths present in the sample prior to circularization. For example, one or more of the mean, median, mode, and standard deviation of fragment lengths in a sample prior to circularization and of circularized polynucleotides is 75%, 80%, 85%, 90%, 95%, or within more.

In some cases, rather than preferentially forming self-associating circularized products, one or more adapters are used such that the 5' and 3' ends of polynucleotides in the sample are joined by one or more intervening adapter oligonucleotides to form circular polynucleotides. Oligonucleotides are used. For example, the 5' end of a polynucleotide can be linked to the 3' end of an adapter, and the 5' end of the same adapter can be linked to the 3' end of the same polynucleotide. Adapter oligonucleotides include any oligonucleotide, at least some of which have a known sequence, that can be linked to a sample polynucleotide. Adapter oligonucleotides can include DNA, RNA, nucleotide analogues, non-canonical nucleotides, labeled nucleotides, modified nucleotides, or combinations thereof. Adapter oligonucleotides can be single-stranded, double-stranded, or partially duplex. Generally, partial duplex adapters include one or more single-stranded regions and one or more double-stranded regions. A double-stranded adapter may comprise two separate oligonucleotides hybridized to each other (also referred to as an "oligonucleotide duplex"), wherein the hybridization is at least one blunt end, one or more 3' overhangs, one or more 5' overhangs, a miss One or more bulges resulting from matched and/or unpaired nucleotides, or any combination thereof, may be left. When two hybridized regions of an adapter are separated from each other by a non-hybridized region, a “bubble” structure results. Different types of adapters, eg adapters of different sequences, may be used in combination. Different adapters can be linked to the sample polynucleotide in a sequential reaction or simultaneously. In some embodiments, identical adapters are added to both ends of a target polynucleotide. For example, a first and second adapter may be added to the same reaction. Adapters can be engineered prior to combining with sample polynucleotides. For example, a terminal phosphate may be added or removed.

When adapter oligonucleotides are used, the adapter oligonucleotides may contain one or more of a variety of sequence elements, including but not limited to one or more amplification primer annealing sequences or their complement, one or more sequencing primer annealing sequences or their complement, one or more A barcode sequence, one or more consensus sequences shared between multiple different adapters or subsets of different adapters, one or more restriction enzyme recognition sites, one or more overhangs complementary to one or more target polynucleotide overhangs, one or more probe binding sites (e.g., , for attachment to a sequencing platform, such as a flow cell for massively parallel sequencing, such as a flow cell as developed by Illumina, Inc.), one or more random or near-random sequences (e.g. , one or more nucleotides randomly selected from the set of two or more different nucleotides at one or more positions, each different nucleotide selected at one or more positions appearing in a set of adapters comprising a random sequence), and combinations thereof. In some cases, the adapter is a bead coated with an oligonucleotide comprising a sequence complementary to the adapter (particularly a tractable magnetic beads) to purify the prototypes containing adapters, wash the prototypes free of adapters and any unligated components, and then release the captured prototypes from the beads. Also, in some cases, complexes of hybridized capture probes and target prototypes can be used directly to generate concatemers, such as by direct rolling circle amplification (RCA). In some embodiments, adapters in circles can also be used as sequencing primers. Two or more sequence elements may be non-adjacent to each other (eg, separated by one or more nucleotides), adjacent to each other, partially overlapping, or completely overlapping. For example, an amplification primer annealing sequence can also serve as a sequencing primer annealing sequence. Sequence elements may be located at or near the 3' end, at or near the 5' end, or internal to an adapter oligonucleotide. A sequence element can be of any suitable length, such as up to about 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or fewer nucleotides in length. there is. Adapter oligonucleotides may be of any suitable length, at least sufficient to accommodate the one or more sequence elements of which they are composed. In some embodiments, an adapter is no more than about 200, 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10 or less nucleotides in length. . In some embodiments, adapter oligonucleotides range from about 12 to 40 nucleotides in length, such as from about 15 to 35 nucleotides in length.

In some embodiments, adapter oligonucleotides linked to fragmented polynucleotides from one sample comprise at least one sequence common to all adapter oligonucleotides and a barcode unique to the adapter linked to polynucleotides from that particular sample, whereby the barcode sequence can be used to distinguish polynucleotides originating from one sample or adapter ligation reaction from polynucleotides originating from another sample or adapter ligation reaction. In some embodiments, an adapter oligonucleotide comprises a 5' overhang, a 3' overhang, or both complementary to one or more target polynucleotide overhangs. Complementary overhangs can be one or more nucleotides in length, including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides in length. there is. Complementary overhangs can include fixed sequences. The complementary overhang of the adapter oligonucleotide may comprise a random sequence of one or more nucleotides, such that one or more nucleotides are randomly selected from a set of two or more different nucleotides at one or more positions, and each of the different nucleotides comprises a random sequence. It is selected at one or more positions shown in the set of adapters with complementary overhangs comprising the sequence. In some embodiments, an adapter overhang is complementary to a target polynucleotide overhang created by restriction endonuclease digestion. In some embodiments, the adapter overhang consists of adenine or thymine.

A variety of methods for circularizing polynucleotides are available. In some embodiments, circularization comprises an enzymatic reaction, such as the use of a ligase (eg, RNA or DNA ligase). A variety of ligases are available, including but not limited to CircLigase™ (Epicentre; Madison, WI), RNA ligase, T4 RNA ligase 1 (ssRNA ligase that acts on both DNA and RNA). In addition, T4 DNA ligase can also ligate ssDNA in the absence of a dsDNA template, but this reaction is generally slow. Other non-limiting examples of ligases are Taq DNA ligase, Thermus filiformis DNA ligase, Escherichia coli DNA ligase, Tth DNA ligase, Thermus scotoductus DNA ligases (I and II), thermostable ligase, Ampligase thermostable DNA ligase, VanC-type ligase, 9°N DNA ligase, Tsp DNA ligase, and bioprospecting NAD-dependent ligases, including discovered novel ligases; T4 RNA ligase, T4 DNA ligase, T3 DNA ligase, T7 DNA ligase, Pfu DNA ligase, DNA ligase 1, DNA ligase III, DNA ligase IV, and novel ligases discovered by bioprospecting ATP-dependent ligase including; and wild-type, mutant subtypes, and genetically engineered variants thereof. When self-association is desired, the concentrations of polynucleotide and enzyme can be adjusted to facilitate the formation of intramolecular circles rather than intermolecular structures. The reaction temperature and time can also be controlled. In some embodiments, 60° C. is used to facilitate intramolecular circularity. In some embodiments, the reaction time is 12 hours to 16 hours. Reaction conditions may be those specified by the manufacturer of the selected enzyme. In some embodiments, an exonuclease step may be included to cleave any unligated nucleic acids after the circularization reaction. That is, the closed circle does not contain a free 5' or 3' end, and thus introduction of a 5' or 3' exonuclease will not cleave the closed circle but will cleave the unligated components. These can be particularly useful in multiplex systems.

Generally, joining the ends of polynucleotides together to form a circular polynucleotide (either directly or using one or more intermediate adapter oligonucleotides) creates a junction having a junction sequence. When the 5' end and 3' end of a polynucleotide are joined via an adapter polynucleotide, the term "junction" can refer to the junction between the polynucleotide and the adapter (e.g., a 5' end junction or a 3' end junction). one of the junctions), the junction between the 5' end and the 3' end of a polynucleotide, such as formed by and including an adapter polynucleotide. Where the 5' and 3' ends of a polynucleotide are joined without intervening adapters (e.g., the 5' and 3' ends of single-stranded DNA), the term "junction" refers to the point at which these two ends are joined. do. A junction can be identified by a sequence of nucleotides comprising the junction (also referred to as “junction sequence”). In some embodiments, the sample is further processed during natural degradation processes (e.g., cell lysis, cell death, and release of DNA from cells into its surroundings, e.g., cell-free polynucleotides, e.g., cell-free DNA and cell-free RNA). fragmentation that is a by-product of sample processing (e.g., fixation, staining, and/or storage), and fragmentation by methods that cut DNA without restriction to a specific target sequence (e.g., other processes that may be degraded). mechanical fragmentation, such as by sonication; non-sequence specific nuclease treatment, such as DNase I, a fragmentase). If the sample contains polynucleotides with a mixture of ends, it is unlikely that two polynucleotides will have the same 5' end or 3' end, and the two polynucleotides will independently have both the same 5' and 3' ends. The chances of having them all are extremely low. Thus, in some embodiments, junctions can be used to distinguish different polynucleotides, even when the two polynucleotides contain a portion with the same target sequence. Where polynucleotide ends are ligated without intervening adapters, junction sequences can be identified by alignment to a reference sequence. For example, if the order of two component sequences appears to be reversed relative to the reference sequence, the point at which the inversion appears to occur may indicate a junction at that point. When polynucleotide ends are joined via one or more adapter sequences, the junctions are either by proximity to known adapter sequences, or sequences from both the 5' and 3' ends of the polynucleotide to which the sequencing reads were circularized as above. can be checked by alignment if it is of sufficient length to obtain In some embodiments, the formation of a particular junction is a sufficiently rare event that it is unique among the circularized polynucleotides of the sample.

Sequencing method

According to some embodiments, linear and/or circularized polynucleotides (or amplification products thereof, which may optionally be enriched) are subjected to a sequencing reaction to generate sequencing reads. Sequencing reads produced by these methods can be used according to other methods disclosed herein. A variety of sequencing methods are available, particularly high-throughput sequencing methods. Examples include, but are not limited to, sequencing systems manufactured by Illumina (sequencing systems such as HiSeq® and MiSeq®), Life Technologies (Ion Torrent®, SOLiD®, etc.), Roche's 454 Life Sciences systems, Pacific Biosciences systems, Oxford Nanopore Technologies , nanoball sequencing, sequencing by hybridization, polymerized colony (POLONY) sequencing, nanogrid rolling circle sequencing (ROLONY), and the like. In some embodiments, sequencing comprises the use of HiSeq® and MiSeq® to produce reads of about or at least about 50, 75, 100, 125, 150, 175, 200, 250, 300 or more nucleotides in length. In some embodiments, sequencing comprises sequencing by synthetic process, wherein individual nucleotides are repeatedly identified as they are added to a growing primer extension product. Pyrosequencing is an example of a sequence by which the incorporation of a nucleotide is confirmed by assaying the resulting synthetic mixture for the presence of a by-product of the sequencing reaction, pyrophosphate. In particular, the primer/template/polymerase complex is contacted with a single type of nucleotide. When these nucleotides are incorporated, the polymerization reaction cleaves the nucleoside triphosphate between the α and β phosphates of the triphosphate chain, releasing pyrophosphate. The presence of the released pyrophosphate is then confirmed using a chemiluminescent enzyme reporter system that converts the pyrophosphate to ATP with AMP and then measures the ATP using a luciferase enzyme to produce a measurable light signal. The base is incorporated when light is detected, and the base is not incorporated when light is not detected. After appropriate washing steps, various bases are periodically contacted with the complex to sequentially identify subsequent bases in the template sequence. See, eg, US Patent No. 6,210,891.

In a related sequencing process, a primer/template/polymerase complex is immobilized on a substrate, and the complex is contacted with labeled nucleotides. Immobilization of the complexes may be achieved through primer sequences, template sequences and/or polymerase enzymes, and may be covalent or non-covalent. For example, immobilization of the complex can be achieved through linkage between the polymerase or primer and the substrate surface. In an alternative configuration, the nucleotides are provided with or without a removable terminating group. Upon incorporation, the label is coupled to the complex and is detectable. For nucleotides with terminators, all four different nucleotides bearing individually identifiable labels are contacted with the complex. Incorporation of the labeled nucleotide stops extension due to the presence of the terminator and adds a label to the complex, allowing identification of the incorporated nucleotide. Labels and terminators are then removed from the incorporated nucleotides, and after appropriate washing steps, the process is repeated. For non-terminated nucleotides, a single type of labeled nucleotide is added to the complex to determine whether the nucleotide will be incorporated as in pyrosequencing. After removal of labeling groups on nucleotides and appropriate washing steps, a variety of different nucleotides are cycled through the reaction mixture in the same process. See, eg, US Pat. No. 6,833,246, which is incorporated herein by reference in its entirety for all purposes. For example, the Illumina genome analyzer system is based on the technology described in WO 98/44151, wherein DNA molecules are coupled to a sequencing platform (flow cell) via anchor probe binding sites (otherwise referred to as flow cell binding sites) and free It is amplified in situ on the slide. The solid surface on which the DNA molecule is amplified typically comprises a plurality of first and second linked oligonucleotides, i.e., a first linked oligonucleotide complementary to a sequence near or at the end of one of the target polynucleotide and the target polynucleotide. and a second linked oligonucleotide that is complementary to a sequence near or at the other end of the. This arrangement enables bridge amplification, described for example in US20140121116. The DNA molecules are then annealed to sequencing primers and sequenced base-by-base in parallel using a reversible terminator approach. Hybridization of the sequencing primers follows cleavage of one strand of the double-stranded bridge polynucleotide at the cleavage site in one of the linked oligonucleotides anchoring the bridge, thus leaving unbound solid substrates that can be removed by denaturation. leaving one single strand and the other strand bound and available for hybridization to the sequencing primer. Typically, the Illumina Genome Analyzer system uses a flow cell with 8 channels to generate sequencing reads between 18 and 36 bases in length and produces high quality data of >1.3 Gbp per run (see www.illumina.com).

In yet another additional sequence by synthetic process, incorporation of differently labeled nucleotides is observed in real time as template dependent synthesis is performed. Individual immobilized primer/template/polymerase complexes can be observed as fluorescently labeled nucleotides are incorporated, allowing real-time identification of each added base as it is added. In this process, a labeling group can be attached to a portion of a nucleotide and cleaved during incorporation. For example, by attaching a label group to a portion of the phosphate chain removed during incorporation, i.e., a β, γ, or other terminal phosphate group on a nucleoside polyphosphate, the label is not incorporated into the resulting strand and instead natural DNA is produced do. Observation of individual molecules may involve optical confinement of the complex within a very small illumination volume. By light-focusing the complex, a monitored region can be created in which randomly diffusing nucleotides can exist for very short periods of time, whereas incorporated nucleotides can remain within the observation volume for longer periods of time when they are incorporated. there is. This can also result in a characteristic signal associated with an incorporation event characterized by a signal profile characteristic of the added base. An interacting label component, such as a pair of fluorescence resonance energy transfer (FRET) dyes, is provided to the polymerase or other part of the complex and the incorporating nucleotide, whereby the incorporation event brings the label components into mutual proximity and, in turn, also incorporates them. causes a characteristic signal that is characteristic of a base that is and 7,416,844; and US 20070134128).

In some embodiments, nucleic acids in a sample can be sequenced by ligation. These methods typically use DNA ligase enzymes to identify target sequences, such as, for example, in the polony method and for use in SOLiD technology (Applied Biosystems, now Invitrogen). In general, a population of all possible oligonucleotides of fixed length is provided and labeled according to the sequenced position. Oligonucleotides are annealed and ligated; Preferential ligation by DNA ligase to the matching sequence results in a signal corresponding to the complementary sequence at that position.

The sequencing methods of the present disclosure may provide useful information for a variety of applications, such as, for example, identifying a disease (eg, cancer) in a subject or determining whether a subject is at risk of having (or developing) a disease. there is. Sequencing can provide the sequence of a polymorphic region. Sequencing can provide the length of a polynucleotide, such as DNA (eg, cfDNA). Additionally, sequencing can provide sequences of ends or breakpoints of DNA, such as cfDNA. Sequencing can provide sequences of the borders of protein binding sites or the borders of DNase hypersensitive sites.

Sample

In some embodiments of the various methods described herein, the sample is from a subject. The subject can be any animal, including but not limited to cows, pigs, mice, rats, chickens, cats, dogs, etc., and is generally a mammal such as a human. A sample polynucleotide is often isolated from a cell-free sample from a subject, such as, for example, a blood sample, or a tissue sample, including a fluid sample (eg, saliva) containing nucleic acids, a bodily fluid sample, or an organ sample. . In some cases, the sample is treated to remove cells, or polynucleotides are isolated without a cell extraction step (eg, to isolate cell-free polynucleotides, such as cell-free DNA). Other examples of sample sources include those from blood, urine, feces, nostrils, lungs, intestines, other bodily fluids or excretions, materials derived therefrom, or combinations thereof. In some embodiments, the sample is a blood sample or a portion thereof (eg, blood plasma or serum). Serum and plasma may be of particular interest due to their relative enrichment for tumor DNA associated with higher rates of malignant cell death among these tissues. In some embodiments, a sample from a single individual is divided into multiple distinct samples (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more distinct samples), which is Assays of 2, 3, 4 or more are independently subjected to the methods of the present disclosure. Where the sample is from a subject, the reference sequence may also be derived from the subject, such as a consensus sequence from a sample under analysis or a sequence of a polynucleotide from another sample or tissue of the same subject. For example, a blood sample can be analyzed for ctDNA mutations, while cellular DNA from another sample (eg, oral or skin sample) is analyzed to determine a reference sequence.

Polynucleotides can be extracted from a sample according to any suitable method. A variety of kits are available for extraction of polynucleotides, the choice of which may depend on the type of sample or type of nucleic acid to be isolated. Examples of extraction methods are provided herein, such as those described for any of the various aspects disclosed herein. In one example, the sample may be a blood sample, such as a sample collected in an EDTA tube (eg, BD Vacutainer). Plasma can be separated from peripheral blood cells by centrifugation (eg, 10 minutes at 1900xg at 4°C). Plasma separation performed in this manner on a 6 mL blood sample will typically yield between 2.5 mL and 3 mL of plasma. Circulating cell-free DNA can be extracted from plasma samples using, for example, the QIAmp Circulating Nucleic Acid Kit (Qiagene) according to the manufacturer's protocol. Afterwards, DNA can be quantified (eg on an Agilent 2100 Bioanalyzer using a High Sensitivity DNA kit (Agilent)). As an example, yields of circulating DNA from such plasma samples from healthy individuals can range from 1 ng to 10 ng per mL plasma, and significantly more in diseased (eg, cancer) patient samples.

In some embodiments, the plurality of polynucleotides comprises cell-free polynucleotides, such as cell-free DNA (cfDNA), cell-free RNA (cfRNA), circulating tumor DNA (ctDNA), or circulating tumor RNA (ctRNA). Cell-free DNA circulates in both healthy and diseased individuals. Cell-free RNA circulates in both healthy and diseased individuals. cfDNA (ctDNA) from tumors is not limited to any particular cancer type, but appears to be a common finding across different malignancies. According to some measurements, the free circulating DNA concentration in plasma is about 14 ng/ml to 18 ng/ml in control subjects and about 180 ng/ml to 318 ng/ml in neoplasia patients. Apoptotic and necrotic cell death contributes to cell-free circulating DNA in body fluids. For example, significantly increased circulating DNA levels have been observed in the plasma of patients with prostate cancer and other prostate diseases such as benign prostatic hyperplasia and prostatitis. Circulating tumor DNA is also present in fluids from the organ in which the primary tumor develops. Thus, breast cancer detection can be achieved in ductal lavage; Colorectal cancer detection can be achieved in feces; Lung cancer detection can be achieved in sputum; Prostate cancer detection can be achieved in urine or ejaculate. Cell-free DNA can be obtained from a variety of sources. One common source is a subject's blood sample. However, cfDNA or other fragmented DNA can be derived from a variety of other sources. For example, urine and feces samples can be a source of cfDNA including ctDNA. Cell-free RNA can be obtained from a variety of sources.

In some embodiments, the polynucleotide is subjected to subsequent steps (eg, circularization and amplification) without an extraction step and/or without a purification step. For example, a fluid sample can be treated to remove cells without an extraction step to produce a purified liquid sample and a cell sample, and then DNA is isolated from the purified fluid sample. A variety of procedures are available for isolation of polynucleotides, such as precipitation or non-specific binding to a substrate followed by washing the substrate to release the bound polynucleotide. When polynucleotides are isolated from a sample without a cell extraction step, the polynucleotides will primarily be extracellular or "cell-free" polynucleotides. For example, cell-free polynucleotides may include cell-free DNA (also referred to as “circulating” DNA). In some embodiments, the circulating DNA is circulating tumor DNA (ctDNA) from tumor cells such as bodily fluids or feces (eg, blood samples). Cell-free polynucleotides may include cell-free RNA (also called "circulating" RNA). In some embodiments, the circulating RNA is a circulating tumor RNA (ctRNA) from a tumor cell. Tumors can exhibit apoptosis or necrosis such that tumor nucleic acids are released into the body, including the subject's bloodstream, in different forms and at different levels through a variety of mechanisms. Typically, the size of ctDNA may range from higher concentrations of smaller fragments, generally 70 to 200 nucleotides in length, to lower concentrations of large fragments up to several thousand kilobases.

cancer

The methods herein provide detection of cancer or detection of risk of cancer. Cancer staging is dependent on the cancer type, with each cancer type having its own classification system. Examples of cancer staging or classification systems are described in more detail below.

In certain aspects, the methods provided herein enable early detection of cancer or detection of non-metastatic cancer. Examples of cancers that can be detected according to the methods disclosed herein include, but are not limited to, acanthoma, acinar cell carcinoma, acoustic neuroma, acromegaly melanoma, acromegaly adenoma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoma Blastous leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, acute myelocytic leukemia, acute promyelocytic leukemia, enamel, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenoid odontogenic Tumor, adrenocortical carcinoma, adult T-cell leukemia, invasive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft tissue sarcoma, ameloblastic fibroma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid Cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, caecal cancer, astrocytoma, atypical malformed rhabdomyomas, basal cell carcinoma, basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini ( Bellini) tubular carcinoma, cholangiocarcinoma, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ, carcinoma of the penis, carcinoma of unknown primary site, carcinosarcoma, Castleman's disease, central nervous system embryonic tumor, cerebellar astrocytoma, brain astrocytoma, uterus Cervical cancer, cholangiocarcinoma, chordoma, chondrosarcoma, chordoma, choriocarcinoma, choroidal plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear-cell tumor , colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, protruding dermatofibrosarcoma, dermoid cyst, connective tissue formation small-cell tumor, diffuse large B-cell lymphoma, germinal insufficiency nerve Epithelial tumor, embryonic carcinoma, endoderm sinus tumor, endometrial cancer, endometrial uterine cancer, endometrioid tumor, enteropathy-associated T-cell lymphoma, ependymoma, ependymcytoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, nasal glioblastoma , Ewing family of tumors, Ewing family sarcoma, Ewing sarcoma, extracranial germ cell tumor, extra-testicular germ cell tumor, extrahepatic cholangiocarcinoma, extramammary Paget's disease, fallopian tube cancer, intrafetal fetus, fibroma, fibrosarcoma, follicle Gastrointestinal lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder cancer, ganglionglioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, sematocyte tumor, gestational choriocarcinoma, gestational choriocarcinoma Sex tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, cerebral glioma, glomerular tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, head and neck cancer, heart cancer , hemangioblastoma, hemangiopericytoma, hemangiosarcoma, hematological malignancy, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast-ovarian cancer syndrome, Hodgkin's lymphoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer , intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, Kaposi's sarcoma, kidney cancer, Klatskin's tumor, Krukenberg tumor, laryngeal cancer , laryngeal cancer, lentigo malignant melanoma, leukemia, leukemia, lip and oral cancer, liposarcoma, lung cancer, corpus luteum, lymphangioma, lymphangiosarcoma, lymphoepithelialoma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, Malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, glioma malignant, mesothelioma malignant, peripheral nerve sheath tumor malignant, malignant rhabdomyomas, malignant triton tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, Mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medulloblastoma, medullary epithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, occult primary metastatic squamous neck carcinoma, metastatic urothelial carcinoma, mixed mule mullerian tumor, monocytic leukemia, oral cancer, myxoid tumor, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplastic disease, myelodysplastic syndrome, myelogenous leukemia, myeloid sarcoma , myeloproliferative disease, myxoma, nasal cancer, nasopharyngeal cancer, nasopharyngeal carcinoma, neoplasia, schwannoma, neuroblastoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-melanoma skin cancer , non-small cell lung cancer, ocular tumor, oligodendrogliocytoma, oligodendroglioma, oncocytoma, optic nerve sheath meningioma, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian cancer, ovarian epithelial cancer, ovarian reproductive Cell tumor, ovarian low malignant potential tumor, Paget's disease of the breast, Pancoast tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, sinus cancer, parathyroid cancer, penile cancer, peripheral vascular epithelial cell Tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineal Blastoma, Granulocytoma, Pituitary Adenoma, Pituitary Tumor, Plasma Cell Neoplasia, Pleuropulmonary Blastoma, Multiple Blastoma, Precursor T-lymphoblastic Lymphoma, Primary Central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, peritoneal pseudomyxoma, rectal cancer, renal cell carcinoma, airway carcinoma associated with the NUT gene on chromosome 15, retina Blastoma, rhabdomyomas, rhabdomyosarcoma, Richter's transformation, sacral coccyx teratoma, salivary gland carcinoma, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary neoplasia, seminoma, serous tumor, Sertoli-Leydig Leydig) cell tumor, sex cord-stromal tumor, Sezary syndrome, signet ring cell carcinoma, skin cancer, small cell carcinoma blue, small cell carcinoma, small cell lung cancer, small cell lymphoma, small bowel cancer, soft tissue sarcoma, somatostatinoma, sooty wart, Spinal Cord Tumor, Spinal Tumor, Splenic Marginal Lymphoma, Squamous Cell Carcinoma, Gastric Cancer, Superficial Dilated Melanoma, Supratentorial Primitive Neuroectodermal Tumor, Superficial Epithelial-stromal Tumor, Synovial Sarcoma, T-Cell Acute Lymphoblastic Leukemia, T -Cell giant granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, terminal lymphoma, testicular cancer, follicular cancer, laryngeal cancer, thymic carcinoma, thymoma, thyroid cancer, renal pelvis and ureter of transitional cell carcinoma, transitional cell carcinoma, urinary tract cancer, urethral cancer, urogenital neoplasia, uterine sarcoma, uveal melanoma, vaginal cancer, Verner Morrison syndrome, wartoid carcinoma, visual pathway glioma, vulvar cancer, Walden Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms' tumor, and combinations thereof.

computer system

The present disclosure provides a computer system programmed to implement the methods of the present disclosure. 1 shows a computer system 201 programmed or otherwise configured to implement the methods of the present disclosure. Computer system 201 may control various aspects of the methods of the present disclosure, such as, for example, a method for determining whether a subject has or is at risk of having a disease (eg, cancer).

Computer system 201 includes a central processing unit (CPU, also herein “processor” and “computer processor”) 205 which can be a single-core or multi-core processor, or multiple processors for parallel processing. Computer system 201 may also include a memory or memory location 210 (eg, random access memory, read-only memory, flash memory), an electronic storage device 215 (eg, hard disk), one or more other systems. and a communication interface 220 (eg, a network adapter) for communicating with and a peripheral device 225, such as a cache, other memory, data storage, and/or electronic display adapter. The memory 210, storage device 215, interface 220, and peripheral device 225 communicate with the CPU 205 through a communication bus (solid line), such as a motherboard. The storage device 215 may be a data storage device (or data storage) for storing data. Computer system 201 may be operatively coupled to a computer network (“network”) 230 with the aid of a communication interface 220 . Network 230 may be the Internet, the Internet and/or extranet, or an intranet and/or extranet in communication with the Internet. In some cases, network 230 is a telecommunications and/or data network. Network 230 may include one or more computer servers that may enable distributed computing, such as cloud computing. Network 230 may in some cases implement a peer-to-peer network with the help of computer system 201, which may allow devices connected to computer system 201 to act as clients or servers.

CPU 205 may execute a machine-readable sequence of instructions, which may be implemented as a program or software. Instructions may be stored in memory locations such as memory 210 . Instructions may be directed to CPU 205, which may subsequently program or otherwise configure CPU 205 to implement the methods of the present disclosure. Examples of tasks performed by CPU 205 may include fetch, decode, execute, and writeback.

CPU 205 may be part of a circuit such as an integrated circuit. One or more other components of system 201 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

The storage device 215 may store files such as drivers, libraries, and stored programs. The storage device 215 may store user data, for example user preferences and user programs. In some cases computer system 201 may include one or more additional data storage devices that are external to computer system 201, such as located on a remote server that communicates with computer system 201 over an intranet or the Internet.

Computer system 201 may communicate with one or more remote computer systems via network 230 . For example, computer system 201 may communicate with a remote computer system of a user (eg, a medical provider or patient). Examples of remote computer systems include personal computers (e.g., portable PCs), slate or tablet PCs (e.g., Apple® iPad, Samsung® Galaxy Tab), phones, smartphones (e.g., Apple® iPhone, Android -Enabled devices, including Blackberry®, or personal digital assistants. A user may access computer system 201 through network 230 .

Methods as described herein may be performed by machine (e.g., computer processor) executable code stored in an electronic storage location of computer system 201, such as, for example, memory 210 or electronic storage device 215. can be implemented Machine-executable code or machine-readable code may be provided in software form. In use, code may be executed by processor 205 . In some cases, the code may be retrieved from storage 215 and stored in memory 210 for quick access by processor 205 . In some circumstances, electronic storage device 215 may be excluded, and machine-executable instructions are stored in memory 210 .

The code may be precompiled and configured for use with a machine having a processor suitable for executing the code, or may be compiled during runtime. The code may be provided in a programming language of choice so that the code can be executed in a precompiled, as-compiled manner.

Aspects of the systems and methods provided herein, such as computer system 201, may be implemented in programming. Various aspects of technology may be considered a “product” or “article of manufacture,” typically in the form of machine (or processor) executable code and/or related data executed or embodied on some kind of machine-readable medium. Machine-executable code may be stored in a memory (eg, read-only memory, random-access memory, flash memory) or an electronic storage device such as a hard disk. A “storage” type medium includes any or all types of memory in a computer, processor, etc., or related modules thereof, such as various semiconductor memories, tape drives, disk drives, etc., which can at any time provide non-transitory storage for software programming. can do. All or part of the Software may be communicated over the Internet or various other telecommunication networks from time to time. For example, such communication may enable loading of software from one computer or processor to another computer or processor, eg, from a management server or host computer to the application server's computer platform. Thus, other types of media that may carry software elements include optical, electrical and electromagnetic waves as used over wired and fiber wired networks and over physical interfaces between local devices over various air-links. A physical element that transmits these waves, such as a wired or wireless link, an optical link, etc., can also be considered as a medium holding software. As used herein, unless limited to a non-transitory tangible "storage" medium, the term computer or machine "readable medium" refers to any medium that participates in providing instructions to a processor for execution. refers to

Accordingly, machine-readable media such as computer-executable code may take many forms, including but not limited to tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media include, for example, any storage devices of any computer(s), such as optical or magnetic disks, such as those that may be used to implement the databases shown in the drawings, and the like. Volatile storage media include dynamic memory, such as the main memory of these computer platforms. Tangible transmission media include coaxial cable; Included are copper wires and optical fibers, including wires that contain buses in computer systems. Carrier-wave transmission media can take the form of electrical or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tapes, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punches Card stock tape, any other physical storage medium with a pattern of holes, RAM, ROM, PROM and EPROM, FLASH-EPROM, any other memory chip or cartridge, carrier waves carrying data or commands, cables carrying such carrier waves, or links, or any other medium from which a computer can read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

The computer system 201 may include or communicate with an electronic display 235 that constitutes, for example, a user interface (UI) 240 for presenting results of the methods of the present disclosure. Examples of UIs include, without limitation, graphical user interfaces (GUIs) and web-based user interfaces.

The methods and systems of this disclosure may be implemented by one or more algorithms. Algorithms may be implemented via software when executed by the central processing unit 205 . The algorithm may be, for example, a trained algorithm (or a trained machine learning algorithm), such as a support vector machine or a neural network.

Example

The following examples are provided for the purpose of illustrating various embodiments of the invention and are not intended to limit the invention in any way. These examples, along with the methods described herein, represent preferred embodiments of the present application, are illustrative, and are not intended to limit the scope of the present invention. Modifications and other uses that fall within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example 1: Methods for selective sequencing

A biological sample is obtained from the subject and cell-free nucleic acids are isolated from the sample. Nucleic acids with methylated sequences are separated from nucleic acids without methylated sequences using antibodies that specifically bind to methylated nucleic acids. Nucleic acids bound to the antibody were purified from the antibody, then circularized and sequenced to obtain sequences of cell-free nucleic acids having methylated sequences in the subject.

Although preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Numerous variations, modifications and substitutions will now be made by those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered by the claims.

Claims (67)

A method of processing a plurality of nucleic acid molecules derived from a cell-free biological sample comprising:
(a) contacting the plurality of nucleic acid molecules or derivatives thereof with a plurality of binding agents to provide a first subset of the plurality of nucleic acid molecules and a second subset of the plurality of nucleic acid molecules coupled to the plurality of binding agents; doing;
(b) separating a first subset of the plurality of nucleic acid molecules coupled to the plurality of binding agents from a second subset of the plurality of nucleic acid molecules;
(c) after (b), circularizing the nucleic acid molecules derived from the first subset of the plurality of nucleic acid molecules to obtain circularized nucleic acid molecules; and
(d) identifying the circularized nucleic acid molecule or derivative thereof
A method for processing a plurality of nucleic acid molecules derived from a cell-free biological sample, comprising: The method of claim 1 , wherein the plurality of nucleic acid molecules comprises deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. 3. The method according to claim 1 or 2, wherein the circularization comprises ligating the 5' end and the 3' end of the nucleic acid molecule to each other. 4. The method of any one of claims 1 to 3, wherein circularization comprises coupling adapters to the 3' end, the 5' end, or both the 5' and 3' ends of the nucleic acid molecule. method. 5. The method of any one of claims 1 to 4, further comprising nucleic acid amplifying the circularized nucleic acid molecule to generate a plurality of amplification products of the circularized nucleic acid molecule, wherein (d) The method comprising identifying the plurality of nucleic acid amplification products. The method of claim 5, wherein the nucleic acid amplification is performed by a polymerase having strand displacement activity. 6. The method of claim 5, wherein the nucleic acid amplification is performed by a polymerase having no strand displacement activity. 6. The method of claim 5, wherein amplifying the nucleic acid comprises contacting the circularized nucleic acid molecule with an amplification reaction mixture comprising random primers. The method of claim 5, wherein the nucleic acid amplification comprises contacting the circularized nucleic acid molecule with an amplification reaction mixture comprising one or more primers, each of which specifically hybridizes to a different target sequence through sequence complementarity. how it would be. 10. The method of any one of claims 1-9, wherein the binding agent comprises an antibody or fragment thereof. 11. The method of claim 10, wherein the antibody or fragment thereof specifically binds to a nucleic acid binding protein. 11. The method of claim 10, wherein the nucleic acid binding protein is a chromatin protein. 11. The method of claim 10, wherein the nucleic acid binding protein is a histone. 11. The method of claim 10, wherein the nucleic acid binding protein is a methyl CpG binding protein. 11. The method of claim 10, wherein the nucleic acid binding protein is a transcription factor. 11. The method of claim 10, wherein the nucleic acid binding protein is an RNA binding protein. 10. The method of any one of claims 1 to 9, wherein the antibody or fragment thereof specifically binds to a nucleic acid sequence. 18. The method of claim 17, wherein the nucleic acid sequence is methylated. 10. The method of any one of claims 1-9, wherein the binding agent comprises a polypeptide or a nucleic acid. 20. The method of claim 19, wherein the polypeptide comprises streptavidin. 19. The method of any one of claims 1 to 18, further comprising determining the size of each cell-free nucleic acid molecule of the plurality of cell-free nucleic acid molecules. 20. The method of any one of claims 1-19, wherein (d) comprises sequencing the circularized nucleic acid molecule or derivative thereof. 23. The method of claim 22, wherein the sequencing is sequencing by synthesis, sequencing by ligation, nanopore sequencing, nanoball sequencing, ion detection, sequencing by hybridization, polymerized colony (POLONY) sequencing, nanogrid rolling circle sequencing (ROLONY) ), and Ion Torrent sequencing. 24. The method of any one of claims 1-23, wherein the cell-free biological sample comprises less than 75 nanograms of nucleic acid. 25. The method of any one of claims 1-24, wherein the cell-free biological sample comprises bodily fluid. 26. The method of claim 25, wherein the bodily fluid is urine, saliva, blood, serum, plasma, tears, sputum, cerebrospinal fluid, synovial fluid, mucus, bile, semen, lymph, amniotic fluid, menstrual fluid, or a combination thereof. 27. The method of any one of claims 1-26, wherein (d) comprises sequencing the circularized nucleic acid molecule or derivative thereof. 28. The method of claim 27, wherein the method determines whether a subject has or is at risk of having a disease by processing the sequence against a plurality of reference sequences to identify the sequence corresponding to at least a subset of the plurality of reference sequences. How to include additional steps. 29. The method of claim 28, wherein the disease is cancer. 30. The method of claim 29, wherein the cancer is colon cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, hepatocellular carcinoma, liver cancer, skin cancer, malignant melanoma, endometrial cancer, esophageal cancer, gastric cancer, ovarian cancer, pancreatic cancer, brain cancer, leukemia, lymphoma, and and is selected from the group consisting of myeloma. 29. The method of claim 28, further comprising generating an electronic report indicating that the subject has or is at risk of having the disease using the sequence identified. 29. The method of claim 28, further comprising providing therapeutic intervention to said subject for a disease using said identified sequence. 29. The method of claim 28, further comprising treating the subject for the disease using the identified sequence. 34. The method of claim 32 or 33, wherein the subject is treated by subjecting the subject to chemotherapy or immunotherapy. 29. The method of claim 28, further comprising monitoring the subject for progression or regression using the sequence identified. The method of claim 1 , wherein in (c) the nucleic acid molecule is coupled to a binding agent of the plurality of binding agents. A method of processing a plurality of nucleic acid molecules derived from a cell-free biological sample comprising:
(a) determining a methylation state of a nucleic acid molecule of the plurality of nucleic acid molecules;
(b) determining the size of the nucleic acid molecule of the plurality of nucleic acid molecules; and
(c) processing (i) the methylation status for the nucleic acid molecule of the plurality of nucleic acid molecules for a first database, and (ii) the size for the nucleic acid molecule of the plurality of nucleic acid molecules for a second database So, confirming the association between at least one disease and the methylation status and the size
A method for processing a plurality of nucleic acid molecules derived from a cell-free biological sample, comprising: 38. The method of claim 37, wherein determining the methylation status comprises sequencing the nucleic acid molecules of the plurality of nucleic acid molecules. 39. The method of claim 38, wherein the sequencing is sequencing by synthesis, sequencing by ligation, nanopore sequencing, nanoball sequencing, ion detection, sequencing by hybridization, polymerized colony (POLONY) sequencing, nanogrid rolling circle sequencing (ROLONY) ) and Ion Torrent sequencing. 38. The method of claim 37, wherein determining the methylation status comprises contacting the nucleic acid molecule with a binding agent that specifically binds to a methylated nucleic acid or derivative thereof. 41. The method of any one of claims 37-40, wherein determining the methylation status comprises:
(a) contacting the plurality of nucleic acid molecules with a plurality of binding agents to provide a first subset of the plurality of nucleic acid molecules and a second subset of the plurality of nucleic acid molecules coupled to the plurality of binding agents;
(b) separating the first subset of the plurality of nucleic acid molecules coupled to the plurality of binding agents from the second subset of the plurality of nucleic acid molecules;
(c) after (b), circularizing the nucleic acid molecules derived from the first subset of the plurality of nucleic acid molecules to obtain circularized nucleic acid molecules; and
(d) identifying the circularized nucleic acid molecule or derivative thereof
A method comprising a. 42. The method of any one of claims 37-41, wherein the binding agent comprises an antibody or fragment thereof. 42. The method of any one of claims 37-41, wherein the binding agent comprises a polypeptide or a nucleic acid. 44. The method of claim 43, wherein the polypeptide comprises streptavidin. 45. The method of any one of claims 37-44, wherein the plurality of nucleic acid molecules comprises deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. 46. The method of claim 41 or 45, wherein circularization comprises ligating the 5' end and the 3' end of the nucleic acid molecule to each other. 47. The method of any one of claims 41-46, wherein circularization comprises coupling adapters to the 3' end, the 5' end, or both the 5' and 3' ends of the nucleic acid molecule. method. 48. The method of any one of claims 41 to 47, further comprising nucleic acid amplifying the circularized nucleic acid molecule to generate a plurality of amplification products of the circularized nucleic acid molecule, wherein (d) The method comprising identifying the plurality of nucleic acid amplification products. 49. The method of claim 48, wherein the nucleic acid amplification is performed by a polymerase having strand displacement activity. 49. The method of claim 48, wherein the nucleic acid amplification is performed by a polymerase that does not have strand displacement activity. 49. The method of claim 48, wherein amplifying the nucleic acid comprises contacting the circularized nucleic acid molecule with an amplification reaction mixture comprising random primers. 49. The method of claim 48, wherein the nucleic acid amplification comprises contacting the circularized nucleic acid molecule with an amplification reaction mixture comprising one or more primers, each of which specifically hybridizes to a different target sequence through sequence complementarity. how it would be. 53. The method of any one of claims 41-52, wherein (d) comprises sequencing the circularized nucleic acid molecule or derivative thereof. 54. The method of claim 53, wherein the sequencing is sequencing by synthesis, sequencing by ligation, nanopore sequencing, nanoball sequencing, ion detection, sequencing by hybridization, polymerized colony (POLONY) sequencing, nanogrid rolling circle sequencing (ROLONY) ) and Ion Torrent sequencing. 55. The method of any one of claims 37-54, wherein the cell-free biological sample comprises less than 75 nanograms of nucleic acid. 56. The method of any one of claims 37-55, wherein the cell-free biological sample comprises bodily fluid. 57. The method of claim 56, wherein the bodily fluid is urine, saliva, blood, serum, plasma, tears, sputum, cerebrospinal fluid, synovial fluid, mucus, bile, semen, lymph, amniotic fluid, menstrual fluid, or combinations thereof. 58. The method of any one of claims 37-57, wherein processing the methylation status relative to a plurality of reference methylation statuses and processing the magnitudes relative to a plurality of reference methylation statuses results in at least one sub-selection of the plurality of reference methylation statuses. determining whether the subject has or is at risk of having the disease by ascertaining the methylation status corresponding to a set and the size corresponding to at least a subset of the reference size. 59. The method of claim 58, wherein the disease is cancer. 60. The method of claim 59, wherein the cancer is colon cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, hepatocellular carcinoma, liver cancer, skin cancer, malignant melanoma, endometrial cancer, esophageal cancer, gastric cancer, ovarian cancer, pancreatic cancer, brain cancer, leukemia, lymphoma, and and is selected from the group consisting of myeloma. 59. The method of claim 58, further comprising generating an electronic report indicating that the subject has or is at risk of having the disease using the identified methylation status and the size. 59. The method of claim 58, further comprising providing a therapeutic intervention to the subject for a disease using the determined methylation status and the size. 59. The method of claim 58, further comprising treating the subject for the disease using the determined methylation status and the size. 64. The method of claim 62 or 63, wherein the subject is treated by subjecting the subject to chemotherapy or immunotherapy. 59. The method of claim 58, further comprising monitoring the subject for progression or regression using the identified methylation status and the size. A method of processing a plurality of nucleic acid molecules derived from a cell-free biological sample of a subject comprising:
(a) contacting the plurality of nucleic acid molecules or derivatives thereof with a plurality of binding agents to provide a first subset of the plurality of nucleic acid molecules and a second subset of the plurality of nucleic acid molecules coupled to the plurality of binding agents; doing;
(b) separating the first subset of the plurality of nucleic acid molecules coupled to the plurality of binding agents from the second subset of the plurality of nucleic acid molecules;
(c) after (b), circularizing the nucleic acid molecules derived from the first subset of the plurality of nucleic acid molecules to obtain a first subset of circularized nucleic acid molecules; and
(d) after (b), circularizing nucleic acid molecules derived from the second subset of the plurality of nucleic acid molecules to obtain a second subset of circularized nucleic acid molecules;
(e) sequencing said first subset of circularized nucleic acid molecules or derivatives thereof to obtain a first size and sequencing said second subset of circularized nucleic acid molecules or derivatives thereof to obtain a second size; step;
(f) comparing the first size and the second size to determine the disease level of the subject
A method of processing a plurality of nucleic acid molecules derived from a cell-free biological sample of a subject, comprising: A method of processing a plurality of nucleic acid molecules derived from a cell-free biological sample of a subject comprising:
(a) contacting the plurality of nucleic acid molecules or derivatives thereof with a plurality of binding agents to provide a first subset of the plurality of nucleic acid molecules and a second subset of the plurality of nucleic acid molecules coupled to the plurality of binding agents; doing;
(b) separating the first subset of the plurality of nucleic acid molecules coupled to the plurality of binding agents from the second subset of the plurality of nucleic acid molecules;
(c) after (b), circularizing the nucleic acid molecules derived from the first subset of the plurality of nucleic acid molecules to obtain a first subset of circularized nucleic acid molecules; and
(d) after (b), circularizing nucleic acid molecules derived from the second subset of the plurality of nucleic acid molecules to obtain a second subset of circularized nucleic acid molecules;
(e) sequencing said first subset of circularized nucleic acid molecules or derivatives thereof to obtain a first final sequence, and sequencing said second subset of circularized nucleic acid molecules or derivatives thereof to obtain a second final sequence obtaining;
(f) comparing the first final sequence and the second final sequence to determine the disease level of the subject.
A method of processing a plurality of nucleic acid molecules derived from a cell-free biological sample of a subject, comprising:

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